Methods and means to modify a plant genome

ABSTRACT

Methods and means are provided to modify in a targeted manner the genome of a plant in close proximity to an existing elite event using a double stranded DNA break inducing enzyme. Also provided are plants, in particular cotton plants showing tolerance to a field dose of at least IX of at least one HPPD inhibitor, and methods for making such plants.

FIELD OF THE INVENTION

The invention relates to the field of agronomy. More particularly, theinvention provides methods and means to introduce a targetedmodification, including insertion, deletion or substitution, at aprecisely localized nucleotide sequence in the genome of a plant using acustom-designed double stranded DNA break inducing enzyme. The inventionfurther relates to a cotton plant cell, plant part, plant, or seedcomprising a chimeric gene comprising a nucleic acid sequence encoding aprotein having HPPD activity, wherein said protein has a tryptophan at aposition corresponding to position 336 of SEQ ID NO: 1, wherein saidprotein provides to said plant tolerance to a field dose of at least 1×of at least one HPPD inhibitor, and methods for making such plants.

BACKGROUND ART

The need to introduce targeted modifications in plant genomes, e.g. toprovide plants with agronomically useful traits such as herbicidetolerance, including the control over the location of integration offoreign DNA in plants has become increasingly important. Several methodshave been developed in an effort to meet this need (for a review seeKumar and Fladung, 2001, Trends in Plant Science, 6, pp 155-159), whichmostly rely on the initial introduction of a double stranded DNA breakat the targeted location via expression of a double strand breakinducing (DSBI) enzyme.

Activation of the target locus and/or repair or donor DNA through theinduction of double stranded DNA breaks (DSB) via rare-cuttingendonucleases, such as I-SceI has been shown to increase the frequencyof homologous recombination by several orders of magnitude. (Puchta etal., 1996, Proc. Natl. Acad. Sci. U.S.A., 93, pp 5055-5060; Chilton andQue, Plant Physiol., 2003; D'Halluin et al. 2008 Plant Biotechnol. J. 6,93-102).

WO96/14408 describes an isolated DNA encoding the enzyme I-SceI. ThisDNA sequence can be incorporated in cloning and expression vectors,transformed cell lines and transgenic animals. The vectors are useful ingene mapping and site-directed insertion of genes.

WO00/46386 describes methods of modifying, repairing, attenuating andinactivating a gene or other chromosomal DNA in a cell through an I-SceIinduced double strand break. Also disclosed are methods of treating orprophylaxis of a genetic disease in an individual in need thereof.Further disclosed are chimeric restriction endonucleases.

WO 2005/049842 describes methods and means to improve targeted DNAinsertion in plants using rare-cleaving “double stranded break” inducing(DSBI) enzymes, as well as improved I-SceI encoding nucleotidesequences.

WO2006/105946 describes a method for the exact exchange in plant cellsand plants of a target DNA sequence for a DNA sequence of interestthrough homologous recombination, whereby the selectable or screenablemarker used during the homologous recombination phase for temporalselection of the gene replacement events can subsequently be removedwithout leaving a foot-print and without resorting to in vitro cultureduring the removal step, employing the therein described method for theremoval of a selected DNA by microspore specific expression of a DSBIrare-cleaving endonuclease.

WO2008/037436 describe variants of the methods and means ofWO2006/105946 wherein the removal step of a selected DNA fragmentinduced by a double stranded break inducing rare cleaving endonucleaseis under control of a germline-specific promoter. Other embodiments ofthe method relied on non-homologous endjoining at one end of the repairDNA and homologous recombination at the other end. WO08/148559 describesvariants of the methods of WO2008/037436, i.e. methods for the exactexchange in eukaryotic cells, such as plant cells, of a target DNAsequence for a DNA sequence of interest through homologousrecombination, whereby the selectable or screenable marker used duringthe homologous recombination phase for temporal selection of the genereplacement events can subsequently be removed without leaving afoot-print employing a method for the removal of a selected DNA flankedby two nucleotide sequences in direct repeats.

WO 2003/004659 discloses recombination systems and a method for removingnucleic acid sequences from the chromosomal DNA of eukaryotic organisms.The invention also relates to transgenic organisms (preferably plants),containing said systems or produced by said method.

WO 2006/032426 discloses improved recombination systems and methods foreliminating maker sequences from the genome of plants. Particulary theinvention is based on use of an expression cassette comprising theparsley ubiquitin promoter, and operably linked thereto a nucleic acidsequence coding for a sequence specific DNA-endonuclease.

U.S. provisional application 61/493,579 and EP11004570.5 describemethods and means to modify in a targeted manner the genome of a cottonplant using a double stranded DNA break inducing enzyme and embryogeniccallus.

In addition, methods have been described which allow the design of rarecleaving endonucleases to alter substrate or sequence-specificity of theenzymes, thus allowing to induce a double stranded break at a locus ofinterest without being dependent on the presence of a recognition sitefor any of the natural rare-cleaving endonucleases. Briefly, chimericrestriction enzymes can be prepared using hybrids between a zinc-fingerdomain designed to recognize a specific nucleotide sequence and thenon-specific DNA-cleavage domain from a natural restriction enzyme, suchas Fold. Such methods have been described e.g. in WO 03/080809,WO94/18313 or WO95/09233 and in Isalan et al., 2001, NatureBiotechnology 19, 656-660; Liu et al. 1997, Proc. Natl. Acad. Sci. USA94, 5525-5530). Another way of producing custom-made meganucleases, byselection from a library of variants, is described in WO2004/067736.Custom made meganucleases or redesigned meganucleases with alteredsequence specificity and DNA-binding affinity may also be obtainedthrough rational design as described in WO2007/047859.

WO2007/049095 describes “LADGLIDADG” homing endonuclease variants havingmutations in two separate subdomains, each binding a distinct part of amodified DNA target half site, such that the endonuclease variant isable to cleave a chimeric DNA target sequence comprising the nucleotidesbound by each subdomain.

WO2007/049156 and WO2007/093836 describe I-CreI homing endonucleasevariants having novel cleavage specificity and uses thereof.

WO2007/047859 describes rationally designed meganucleases with alteredsequence specificity and DNA binding affinity.

WO11/064736 describes optimized endonucleases, as well as methods oftargeted integration, targeted deletion or targeted mutation ofpolynucleotides using optimized endonucleases. WO11/064750 describeschimeric endonucleases, comprising an endonuclease and a heterologousDNA binding domain comprising one or more Zn2C6 zinc fingers, as well asmethods of targeted integration, targeted deletion or targeted mutationof polynucleotides using chimeric endonucleases and WO11/064751describes chimeric endonucleases, comprising an endonuclease and aheterologous DNA binding domain, as well as methods of targetedintegration, targeted deletion or targeted mutation of polynucleotidesusing chimeric endonucleases.

PCT/EP11/002894 and PCT/EP11/002895 describe methods and means to modifyin a targeted manner the plant genome of transgenic plants comprisingchimeric genes wherein the chimeric genes have a DNA element commonlyused in plant molecular biology, as well as re-designed meganucleases tocleave such an element commonly used in plant molecular biology.

WO 2009/006297 discloses methods and compositions for altering thegenome of a monocot plant cell, and a monocot plant, involving the useof a double-stranded break inducing agent to alter a monocot plant orplant cell genomic sequence comprising a recognition sequence for thedouble-stranded break inducing agent.

Gao et al. 2010, The Plant Journal 61, p 176-187 describe heritabletargeted mutagenesis in maize using a re-designed endonuclease.

However, in order to efficiently make combinations of agronomicallyuseful traits without having to resort to elaborate breeding schemes orto test large numbers of single events, there thus still remains a needfor functional re-designed meganucleases which can recognize arecognition site in close proximity to an already existing elite event,and uses thereof in order to make stacks of genes conferringagronomically favorable properties at a single genetic locus.

One of such agronomically useful traits is tolerance to herbicides, suchas HPPD-inhibitor herbicides. HPPD (hydroxyphenylpyruvate dioxygenase)proteins are enzymes which catalyse the reaction in whichpara-hydroxyphenylpyruvate (abbreviated herein as HPP), a tyrosinedegradation product, is transformed into homogentisate (abbreviatedherein as HG), the precursor in plants of tocopherol and plastoquinone(Crouch N. P. et al. (1997) Tetrahedron, 53, 20, 6993-7010, Fritze etal., (2004), Plant Physiology 134:1388-1400). Tocopherol acts as amembrane-associated antioxidant. Plastoquinone, firstly acts as anelectron carrier between photosystem II (PSII) and the cytochrome b6/fcomplex and secondly, is a redox cofactor for phytoene desaturase, whichis involved in the biosynthesis of carotenoids.

Up to now, more than 700 nucleic acid sequences from various organismspresent in NCBI database were annotated as coding for a putative proteinhaving an HPPD domain. Several HPPD proteins and their primary sequenceshave been described in the state of the art, in particular the HPPDs ofbacteria such as Pseudomonas (Rüetschi et al., Eur. J. Biochem., 205,459-466, 1992, WO 96/38567), of plants such as Arabidopsis (WO 96/38567,Genebank AF047834), carrot (WO 96/38567, Genebank 87257), Avena sativa(WO 02/046387), wheat (WO 02/046387), Brachiaria platyphylla (WO02/046387), Cenchrus echinatus (WO 02/046387), Lolium rigidum (WO02/046387), Festuca arundinacea (WO 02/046387), Setaria faberi (WO02/046387), Eleusine indica (WO 02/046387), Sorghum (WO 02/046387),Coccicoides (Genebank COITRP), of Coptis japonica (WO 06/132270),Chlamydomonas reinhardtii (ES 2275365), or of mammals such as mouse orpig.

Inhibition of HPPD leads to uncoupling of photosynthesis, deficiency inaccessory light-harvesting pigments and, most importantly, todestruction of chlorophyll by UV-radiation and reactive oxygen species(bleaching) due to the lack of photo protection normally provided bycarotenoids (Norris et al. (1995), Plant Cell 7: 2139-2149). Bleachingof photosynthetically active tissues leads to growth inhibition andplant death.

Some molecules which inhibit HPPD, and which bind specifically to theenzyme in order to inhibit transformation of the HPP into homogentisate,have proven to be very effective selective herbicides. At present, mostcommercially available HPPD inhibitor herbicides belong to one of thesethree chemical families:

-   -   1) the triketones, e.g. sulcotrione, mesotrione; tembotrione;        tefuryltrione; bicyclopyrone; benzobicyclon    -   2) the isoxazoles, e.g. isoxaflutole, or corresponding        diketonitriles. In plants, isoxazoles such as isoxaflutole are        rapidly converted into diketonitriles, which exhibit the HPPD        inhibitor property; and    -   3) the pyrazolinones, e.g. topramezone, pyrasulfotole and        pyrazoxyfen.

These HPPD-inhibiting herbicides can be used against grass and/or broadleaf weeds in crop plants that display metabolic tolerance, such asmaize (Zea mays) in which they are rapidly degraded (Schulz et al.,1993; Mitchell et al., 2001; Garcia et al., 2000; Pallett et al., 2001).In order to extend the scope of these HPPD-inhibiting herbicides,several efforts have been developed in order to confer to plants,particularly plants without or with an underperforming metabolictolerance, a tolerance level acceptable under agronomic fieldconditions.

In that context, it has first been demonstrated that the mereoverexpression of a native HPPD enzyme in transformed sensitive plantsdoes provide an effective tolerance to HPPD inhibitors to thetransformed plants (WO96/38567).

Another strategy was to mutate the HPPD in order to obtain a targetenzyme which, while retaining its properties of catalysing thetransformation of HPP into homogentisate, is less sensitive to HPPDinhibitors than is the native HPPD before mutation.

This strategy has been successfully applied for the production of plantstolerant to HPPD-inhibitors, by transforming plants with a gene encodingan HPPD enzyme mutated at one or more positions in its C-terminal part(WO 99/24585). Among the useful mutations in the C-terminal part of HPPDenzymes which can confer tolerance to HPPD-inhibitors, certain mutationswere shown to provide increased tolerance to certain diketonitrileherbicides, for example the mutations Pro215Leu, Gly336Glu, Gly336Ile,and Gly336Trp (positions of the mutated amino acid are indicated withreference to the Pseudomonas HPPD).

More recently, it has been shown in patent application WO 2009/144079that certain specific amino acid substitutions at position 336 of theHPPD provide tolerance to certain HPPD inhibitor herbicides in vitro.

US 2010/0197503 also indicates a number of mutations at differentpositions within or close to the active site of the HPPD taken fromAvena sativa and examines some of these mutated HPPD enzymes for theirinhibition by certain HPPD inhibitors such as sulcotrione.

Despite these successes obtained for the development of plants showingtolerance to some HPPD inhibitors herbicides described above, it isstill desirable to develop and/or improve the tolerance of specificplants such as cotton, to more, newer or to several different HPPDinhibitors, particularly HPPD inhibitors belonging to the classes of thetriketones (e.g. sulcotrione, mesotrione, tembotrione, tefuryltrione,bicyclopyrone and benzobicyclon), the pyrazolinones (e.g., topramezone,pyrasulfotole and pyrazoxifen) and the isoxazoles (e.g. isoxaflutole) orcorresponding diketonitriles. This problem is solved as herein afterdescribed in the different embodiments, examples and claims.

These and other problems are solved as described hereinafter in thedifferent detailed embodiments of the invention, as well as in theclaims.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to a method for modifying thegenome of a plant cell at a predefined site comprising the steps of

-   -   a. inducing a double stranded DNA break in the vicinity of or at        said predefined site, said double stranded break being induced        by the introduction into said cell of a double stranded DNA        break inducing (DSBI) enzyme which recognizes a recognition        sequence in the vicinity of or at said predefined site;    -   b. selecting a plant cell wherein said double stranded DNA break        has been repaired resulting in a modification in the genome at        said preselected site, wherein said modification is selected        from        -   i. a replacement of at least one nucleotide;        -   ii. a deletion of at least one nucleotide;        -   iii. an insertion of at least one nucleotide; or        -   iv. any combination of i.-iii.;    -   characterized in that said predefined site and/or recognition        site is located in close proximity to an elite event.

In a particular embodiment the event is GHB119.

In another embodiment, the recognition sequence is comprised within SEQID NO: 3 or SEQ ID NO: 4.

The recognition sequence may comprise the nucleotide sequence of SEQ IDNO. 1 or SEQ ID NO: 2.

The invention further provides a method for modifying the genome of aplant cell at a predefined site comprising the steps of

-   -   a. inducing a double stranded DNA break in the vicinity of or at        said predefined site, said double stranded break being induced        by the introduction into said cell of a double stranded DNA        break inducing (DSBI) enzyme which recognizes a recognition        sequence in the vicinity of or at said predefined site;    -   b. selecting a plant cell wherein said double stranded DNA break        has been repaired resulting in a modification in the genome at        said preselected site, wherein said modification is selected        from        -   i. a replacement of at least one nucleotide;        -   ii. a deletion of at least one nucleotide;        -   iii. an insertion of at least one nucleotide; or        -   iv. any combination of i.-iii.;    -   characterized in that said recognition site comprises the        nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 2.

The DSBI enzyme may be introduced into said cell by the delivery intosaid cell of a nucleic acid molecule comprising one or more chimericgenes encoding (together) said endonuclease enzyme. The DSBI enzyme maybe a single chain meganuclease or a pair of meganucleases whichrecognizes or recognize in concert said predefined site and induces orinduce said double stranded break.

In a particular embodiment, the meganuclease or pair of meganucleasesis/are derived from I-CreI and wherein the following amino acids arepresent in meganuclease unit 1:

-   -   a. S at position 32;    -   b. Y at position 33;    -   c. Q at position 38;    -   d. Q at position 80;    -   e. S at position 40;    -   f. T at position 42;    -   g. R at position 77;    -   h. Y at position 68;    -   i. Q at position 70;    -   j. H at position 75;    -   k. T at position 44;    -   l. I at position 24;    -   m. Q at position 26;    -   n. K at position 28;    -   o. N at position 30.

and wherein the following amino acids are present in meganuclease unit2:

-   -   p. S at position 70;    -   q. Q at position 44;    -   r. K at position 24;    -   s. A at position 26;    -   t. K at position 28;    -   u. N at position 30;    -   v. S at position 32;    -   w. Y at position 33;    -   x. Q at position 38;    -   y. Q at position 80;    -   z. S at position 40;    -   aa. T at position 42;    -   bb. Q at position 77;    -   cc. Y at position 68.

The meganuclease or pair of meganucleases may comprises the amino acidsequence of SEQ ID NO. 6 from amino acid position 11-165 and fromposition 204-360.

The meganuclease or pair of meganucleases may also be encoded by one ormore nucleotide sequences which comprises or comprise together thenucleotide sequences of SEQ ID NO 5 from nucleotide position 3120-3584and from position 3698-4169.

In one embodiment, prior to step b. a repair DNA molecule is deliveredinto said cell, said repair DNA molecule being used as a template forrepair of said double stranded DNA break.

The repair DNA may comprise at least one flanking region comprising anucleotide sequence having sufficient homology to the upstream ordownstream DNA region of said predefined site to allow recombinationwith said upstream or downstream DNA region. Alternatively, the repairDNA may comprise two flanking regions located on opposite ends of saidrepair DNA, one of said flanking regions comprising a nucleotidesequence having sufficient homology to the upstream DNA region of saidpredefined site, the other flanking region comprising a nucleotidesequence having sufficient homology to the downstream sequence of saidpredefined site to allow recombination between said flanking nucleotidesequences and said upstream and downstream DNA regions.

In a further embodiment, the repair DNA comprises a selectable markergene and/or a plant expressible gene of interest. The plant expressiblegene of interest can be selected from the group of a herbicide tolerancegene, an insect resistance gene, a disease resistance gene, an abioticstress resistance gene, an enzyme involved in oil biosynthesis,carbohydrate biosynthesis, an enzyme involved in fiber strength or fiberlength, an enzyme involved in biosynthesis of secondary metabolites.

In yet another embodiment, plant cell of which the genome was modifiedat a predefined position is further regenerated into a plant, which alsocontains the modification at the predefined position. That plant canthen be crossed with another plant, resulting in offspring alsocomprising the genomic modification.

The invention further relates to a plant cell comprising a modificationat a predefined site of the genome, obtained by the method as describedabove. Also encompassed within the invention are; a plant, plant part,seed or propagating material thereof, comprising a modification at apredefined site of the genome, obtained by the method of the inventionor consisting essentially of the plant cells of the invention.

Also provided is a method of growing a plant of the invention, i.e. aplant comprising a modification at a predefined site of the genome,comprising the step of applying a chemical to said plant or substratewherein said plant is grown, as well as a method for producing a plantcomprising a modification at a predefined site of the genome, comprisingthe step of crossing a plant consisting essentially of the plant cellsof the invention or a plant of the invention (plant cells and plantscomprising the intended genomic modification) with another plant or withitself and optionally harvesting seeds.

In one embodiment, the invention also provides a cotton plant cell,plant part, plant, or seed comprising a chimeric gene comprising

-   -   (a) a nucleic acid sequence encoding a protein having HPPD        activity, wherein said protein has a tryptophan at a position        corresponding to position 336 of SEQ ID NO: 19, wherein said        protein provides to said plant tolerance to a field dose of at        least 1× of at least one HPPD inhibitor, operably linked to    -   (b) a plant expressible promoter and optionally    -   (c) a translational termination and polyadenylation region.

The protein having HPPD activity may have at least 95% sequence identityto the amino acid sequence of SEQ ID NO: 21. The nucleic acid sequenceencoding the protein having HPPD activity may be optimized forexpression in cotton. The protein may comprise the amino acid sequenceof SEQ ID NO: 21 or may be encoded by a nucleic acid comprising thenucleotide sequence of SEQ ID NO: 20 from nt 949 to nt 2025.

The cotton plant cell, plant part, plant, or seed may have a toleranceto a field dose of at least 1.5× of said at least one HPPD inhibitor, orto a field dose of at least 2× of said at least one HPPD inhibitor, orto a field dose of at least 4× of said at least one HPPD inhibitor.

The at least one HPPD inhibitor may be selected from mesotrione,isoxaflutole, topramezone, pyrasulfutole and tembotrione.

The cotton plant cell, plant part, plant, or seed of the invention maybe tolerant to at least two HPPD inhibitors, preferably at least threeHPPD inhibitors, more preferably at least four HPPD inhibitors such asat least 5 or at least 6 HPPD inhibitors.

The chimeric gene of the cotton plant cell, plant part, plant, or seedaccording to the invention may comprise the nucleic acid sequence of SEQID NO: 20 from position 88 to position 2714.

The cotton plant cell, plant part, plant or seed of the invention mayalso comprises at least one further chimeric gene comprising a nucleicacid sequence encoding an enzyme providing to the plant tolerance to aherbicide which is not an HPPD inhibitor or providing tolerance to atleast one insect or fungal species.

In another embodiment, the invention provides a method for obtaining acotton plant or plant cell tolerant to field dose of at least 1× of atleast one HPPD inhibitor, comprising

-   -   introducing a chimeric gene comprising:    -   (a) a nucleic acid sequence encoding a protein having HPPD        activity, wherein said protein has a tryptophan at a position        corresponding to position 336 of SEQ ID NO: 19, wherein said        protein provides to said plant tolerance to a field dose of at        least 1× of at least one HPPD inhibitor, operably linked to    -   (b) a plant expressible promoter and optionally    -   (c) a translational termination and polyadenylation region.

Also provided is a method for controlling weeds in the vicinity of acotton plant or on a plant field comprising

-   -   applying at least one HPPD inhibitor to the vicinity of a cotton        plant or to a cotton plant field in a field dose of at least 1×.

In the methods according to the invention the at least one HPPDinhibitor may be selected from mesotrione, isoxaflutole, topramezone,pyrasulfutole and tembotrione. The at least one HPPD inhibitor isapplied in a field dose of at least 1.5×, at least 2× or at least 4×.

In a particular embodiment of the methods of the invention the at leastone HPPD inhibitor is isoxaflutole and mesotrione, wherein saidisofluxatole is applied pre-emergence and said mesotrione is appliedpost-emergence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Schematic representation of the recognition site andinteractions with amino acids of the different meganuclease monomericunits COT5 and COTE, represented by SEQ ID No. 1 and SEQ ID No. 2.

FIG. 2: Amino acid sequence of the single chain COT-5/6 meganuclease,comprising an SV40 nuclear localization signal (amino acids 1-10), theCOT-5 subunit (amino acid 11-165), a linker sequence (amino acids166-203) and the COT-6 subunit (amino acids 204-360), represented by SEQID No. 6.

FIG. 3: Schematic overview of targeted insertion through at leastone-sided homologous recombination. Scissors indicate recognition sitesfor DSBI enzymes (COT-5/6), block arrows represent promoters, smallrectangles represent terminators, while large rectangles representgenes, small arrows represent PCR primers, double-headed arrowsrepresent southern blotting fragments, vertical block arrows representrestriction sites, dotted lines represent flanking DNA sequences inwhich homologous regions are indicated by the accolades. Repair DNAvector pCV211 comprises a 2mEPSPS gene and an HPPD-336W flanked byflanking regions with homology to the regions surrounding the COT-5/6recognition site in the 3′ flanking sequence of the GHB119 elite event.After double stranded DNA break induction by COT-5/6, homologousrecombination between the 1561 by and 2072 by flanking regions of thepCV211 vector with the corresponding regions in the 3′ flanking sequenceof GHB119, the 2mEPSPS gene and HPPD-336W genes of pCV211 are insertedat the target site. A correctly stacked event can be identified by PCRusing primer pair IB617×IB572 and IB616×IB572 and by the identificationof an 11 kb genomic band by southern blotting on DraIII/StuI digestedgenomic DNA with probes directed to EPSPS, HPPD or Cry2Ae.

DESCRIPTION OF DIFFERENT EMBODIMENTS OF THE INVENTION

The current invention is based on the observation that functionalre-designed meganucleases can be obtained which specifically recognizeand cleave a nucleotide sequence (SEQ ID No. 1 and SEQ ID No. 2—FIG. 1),which nucleotide sequence is present in close proximity of an existingevent, namely cotton event GHB119 (described in 2008/151780, deposit nrATCC PTA-8398), comprising the Cry2AE gene and the bar gene (conferringLepidoptera resistance and glufosinate tolerance respectively). Usingthis specific meganuclease an additional DNA fragment comprising anEPSPS gene and an HPPD gene (conferring tolerance to glyphosate and HPPDinhibiting herbicides respectively) could be inserted within a few kb ofthe existing GHB119 event, thereby creating a quadruple gene stack whichshould inherit as a single genetic unit.

A cotton plant was thus generated comprising a chimeric DNA moleculeencoding a protein having HPPD activity in which the conserved aminoacid corresponding to glycine (Gly or G) at a position corresponding toposition 336 of the Pseudomonas fluorescens HPPD protein has beenreplaced by a tryptophan (Trp or W).

It was furthermore surprisingly found that plants, particularly cottonplants, comprising such a chimeric gene leading to the expression of aprotein having HPPD activity having a mutation to Trp instead of theconserved native amino acid residue Gly at the position corresponding toposition 336 in the amino acid sequence of the protein of Pseudomonasfluorescens, whether as a quadruple stack (i.e. targeted) or whethergenerated by random transformation, showed tolerance to a field dose ofat least 1× of several HPPD inhibitor herbicides, such as mesotrione,isoxaflutole, tembotrione, pyrasulfotole and topramezone or any otherapplicable HPPD inhibitor as listed herein. Surprisingly, the presentinventors found that plants, in particular cotton plants, expressing aprotein having HPPD activity having a mutation to Trp instead of theconserved native amino acid residue Gly at the position corresponding toposition 336 in the amino acid sequence of the protein of Pseudomonasfluorescens, showed tolerance to a field dose of at least 1× of severalHPPD inhibitors.

There are several advantages of being able to introduce a genomicmodification in close proximity to an existing elite event. Firstly, themodification will co-segregate with the earlier event, thereby avoidingcomplex breeding schemes normally required to combine certain traitsconferred by single events. Secondly, the modification will occur in afavorable genomic environment for expression of the desired traitresulting from the modification as the existing elite event is alsoassumed to show a correct, appropriate and stable spatial and temporalphenotypic expression due to its particular genomic localization, aselaborated below.

Accordingly, in one embodiment, the invention relates to a method formodifying the genome of a plant cell at a predefined site comprising thesteps of

-   -   a. inducing a double stranded DNA break in the vicinity of or at        said predefined site, said double stranded break being induced        by the introduction into said cell of an double stranded DNA        break inducing (DSBI) enzyme which recognizes a recognition        sequence in the vicinity of or at said predefined site;    -   b. selecting a plant cell wherein said double stranded DNA break        has been repaired resulting in a modification in the genome at        said preselected site, wherein said modification is selected        from        -   v. a replacement of at least one nucleotide;        -   vi. a deletion of at least one nucleotide;        -   vii. an insertion of at least one nucleotide; or        -   viii. any combination of i.-ii.;    -   characterized in that said predefined site and/or said        recognition sequence is/are located in close proximity to an        existing elite event.

As used herein, a “double stranded DNA break inducing rare-cleavingendonuclease” is an enzyme capable of inducing a double stranded DNAbreak at a particular nucleotide sequence, called the “recognitionsite”. Rare-cleaving endonucleases are rare-cleaving in the sense thatdue to their long recognition sequence (typically about 14-40 nt) theyhave a very low frequency of cleaving, even in the larger plant genomes,e.g. they cut only 10 times, only 5 times, only 4 times, only threetimes, only two times or only once per genome. Homing endonucleasesconstitute a family of such rare-cleaving endonucleases and aresometimes also referred to as meganuclease. They may be encoded byintrons, independent genes or intervening sequences, and presentstriking structural and functional properties that distinguish them fromthe more classical restriction enzymes, usually from bacterialrestriction-modification Type II systems. Their recognition sites have ageneral asymmetry which contrast to the characteristic dyad symmetry ofmost restriction enzyme recognition sites. Several homing endonucleasesencoded by introns or inteins have been shown to promote the homing oftheir respective genetic elements into allelic intronless or inteinlesssites. By making a site-specific double strand break in the intronlessor inteinless alleles, these nucleases create recombinogenic ends, whichengage in a gene conversion process that duplicates the coding sequenceand leads to the insertion of an intron or an intervening sequence atthe DNA level.

A well characterized horning endonuclease is I-SceI. I-SceI is asite-specific endonuclease, responsible for intron mobility inmitochondria in Saccharomyces cerevisea. The enzyme is encoded by theoptional intron Sc LSU.1 of the 21S rRNA gene and initiates a doublestranded DNA break at the intron insertion site generating a 4 bystaggered cut with 3′OH overhangs. The recognition site of I-SceIendonuclease extends over an 18 by non-symmetrical sequence (Colleaux etal. 1988 Proc. Natl. Acad. Sci. USA 85: 6022-6026). The amino acidsequence for I-SceI and a universal code equivalent of the mitochondrialI-SceI gene have been provided by e.g. WO 96/14408. WO 96/14408 furtherdiscloses a number of variants of I-SceI protein which are stillfunctional.

PCT application PCT/EP04/013122 (incorporated herein by reference)provides synthetic nucleotide sequence variants of I-SceI which havebeen optimized for expression in plants.

A list of other rare cleaving DSB inducing enzymes and their respectiverecognition sites is provided in Table I of WO 03/004659 (pages 17 to20) (incorporated herein by reference). These include I-Sce I, I-Chu I,I-Dmo I, I-Cre I, I-Csm I, PI-Fli I, Pt-Mtu I, I-Ceu I, I-Sce II, I-SceIII, HO, PI-Civ I, PI-Ctr I, PI-Aae I, PI-BSU I, PI-DhaI, PI-Dra I,PI-May I, PI-Mch I, PI-Mfu I, PI-Mfl I, PI-Mga I, PI-Mgo I, PI-Min I,PI-Mka I, PI-Mle I, PI-Mma I, PI-Msh I, PI-Msm I, PI-Mth I, PI-Mtu I,PI-Mxe I, PI-Npu I, PI-Pfu I, PI-Rma I, PI-Spb I, PI-Ssp I, PI-Fac I,PI-Mja I, PI-Pho I, PI-Tag I, PI-Thy I, PI-Tko I or PI-Tsp I.

Furthermore, methods are available to design custom-tailoredrare-cleaving endonucleases that recognize basically any targetnucleotide sequence of choice. Briefly, chimeric restriction enzymes canbe prepared using hybrids between a zinc-finger domain designed torecognize a specific nucleotide sequence and the non-specificDNA-cleavage domain from a natural restriction enzyme, such as Fold.These enzymes are generally referred to as Zinc finger endonucleases(ZFEs). Such methods have been described e.g. in WO 03/080809,WO94/18313 or WO95/09233 and in Isalan et al., 2001, NatureBiotechnology 19, 656-660; Liu et al. 1997, Proc. Natl. Acad. Sci. USA94, 5525-5530). Another way of producing custom-made meganucleases, byselection from a library of variants, is described in WO2004/067736.Custom made meganucleases with altered sequence specificity andDNA-binding affinity may also be obtained through rational design asdescribed in WO2007/047859. Another example of custom-designedrare-cleaving endonucleases include the so-called TALE nucleases, whichare based on transcription activator-like effectors (TALEs) from thebacterial genus Xanthomonas fused to the catalytic domain of e.g. FOKI.The DNA binding specificity of these TALEs is defined by repeat-variablediresidues (RVDs) of tandem-arranged 34/35-amino acid repeat units,which can be modified to recognize specific target sequences (Christianet al., 2010, Genetics 186: 757-761, WO10/079430 and WO11/146121. Suchcustom designed endonucleases are also referred to as a non-naturallyoccurring endonucleases.

Since the re-designed meganucleases are derived from naturally occurringendonucleases, the available potential recognition sites are notentirely random but appear to have some degree of resemblance to thenucleotide sequence originally recognized by the naturally occurringendonuclease upon which the re-designed meganuclease is based. As statedby Gao et al (2010, The Plant Journal, pp 1-11) the structure-basedprotein design method to modify the DNA-binding characteristics ofI-CreI is based on visual inspection of the I-CreI-DNA co-crystalstructure leading to a prediction of a a large number of amino acidsubstitutions that change I-CreI base preference at particular positionsin its recognition site. Individual amino acid substitutions wereevaluated experimentally, and those that conferred the desired change inbase preference were added to a database of mutations that can be “mixedand matched” to generate derivatives of I-CreI that recognize highlydivergent DNA sites. In theory, the combinatorial diversity availableusing the current mutation database is sufficient to target anengineered endonuclease approximately every 1000 by in a random DNAsequence.

An “event”, as used herein, is defined as a (artificial) genetic locusthat, as a result of genetic engineering, carries a foreign DNA ortransgene comprising at least one copy of a gene of interest or ofmultiple genes of interest at a particular genomic location. The typicalallelic states of an event are the presence or absence of the foreignDNA. An event is characterized phenotypically by the expression of thetransgene. At the genetic level, an event is part of the genetic make-upof a plant. At the molecular level, an event can be characterized by therestriction map (e.g., as determined by Southern blotting), by theupstream and/or downstream flanking sequences of the transgene(reflecting the genomic location), the location of molecular markersand/or the molecular configuration of the transgene. Usuallytransformation of a plant with a transforming DNA comprising at leastone gene of interest leads to a population of transformants comprising amultitude of separate events, each of which is unique. An event ischaracterized by the foreign DNA and at least one of the flankingsequences.

An elite event, as used herein, is an event which is selected from agroup of events, obtained by transformation with the same transformingDNA, based on the expression and stability of the transgene(s) and thetrait it confers, as well as its compatibility with optimal agronomiccharacteristics of the plant comprising it. Thus the criteria for eliteevent selection are one or more, preferably two or more, advantageouslyall of the following:

a) that the presence of the foreign DNA does not compromise otherdesired characteristics of the plant, such as those relating toagronomic performance or commercial value (e.g. does not cause anincreased susceptibility to disease, does not cause a yield drag, ordoes not cause increased lodging, etc);b) that the event is characterized by a well defined molecularconfiguration which is stably inherited and for which appropriate toolsfor identity control can be developed;c) that the gene(s) of interest show(s) a correct, appropriate andstable spatial and temporal phenotypic expression, both in heterozygous(or hemizygous) and homozygous condition of the event, at a commerciallyacceptable level in a range of environmental conditions in which theplants carrying the event are likely to be exposed in normal agronomicuse.

It is furthermore preferred that the foreign DNA is associated with aposition in the plant genome that allows easy introgression into desiredcommercial genetic backgrounds.

The status of an event as an elite event is confirmed by introgressionof the elite event in different relevant genetic backgrounds andobserving compliance with one, two or all of the criteria e.g. a), b)and c) above.

An “elite event” thus refers to a genetic locus comprising a foreignDNA, which meets the above-described criteria. A plant, plant materialor progeny such as seeds can comprise one or more elite events in itsgenome.

Once one or both of the flanking sequences of the foreign DNA have beensequenced, primers and probes can be developed which specificallyrecognize this (these) sequence(s) in the nucleic acid (DNA or RNA) of asample by way of a molecular biological technique. For instance a PCRmethod can be developed to identify the elite event in biologicalsamples (such as samples of plants, plant material or productscomprising plant material). Such a PCR is based on at least two specific“primers”, one recognizing a sequence within the 5′ or 3′ flankingsequence of the elite event and the other recognizing a sequence withinthe foreign DNA. The primers preferably have a sequence of between 15and 35 nucleotides which under optimized PCR conditions “specificallyrecognize” a sequence within the 5′ or 3′ flanking sequences of theelite event and the foreign DNA of the elite event respectively, so thata specific fragment (“integration fragment” or discriminating amplicon)is amplified from a nucleic acid sample comprising the elite event. Thismeans that only the targeted integration fragment, and no other sequencein the plant genome or foreign DNA, is amplified under optimized PCRconditions.

Transgenic plants containing elite transformation events, or acombination of transformation events, that may be used according to themethods of the invention, include those that are listed for example inthe databases for various national or regional regulatory agencies, andinclude Event 1143-14A (cotton, insect control, not deposited, describedin WO2006/128569); Event 1143-51B (cotton, insect control, notdeposited, described in WO2006/128570); Event 1445 (cotton, herbicidetolerance, not deposited, described in US2002120964 or WO2002/034946);Event 17053 (rice, herbicide tolerance, deposited as PTA-9843, describedin WO2010/117737); Event 17314 (rice, herbicide tolerance, deposited asPTA-9844, described in WO2010/117735); Event 281-24-236 (cotton, insectcontrol—herbicide tolerance, deposited as PTA-6233, described inWO2005/103266 or US2005216969); Event 3006-210-23 (cotton, insectcontrol—herbicide tolerance, deposited as PTA-6233, described inUS2007143876 or WO2005/103266); Event 3272 (corn, quality trait,deposited as PTA-9972, described in WO2006098952 or US2006230473); Event40416 (corn, insect control—herbicide tolerance, deposited as ATCCPTA-11508, described in WO2011/075593); Event 43A47 (corn, insectcontrol—herbicide tolerance, deposited as ATCC PTA-11509, described inWO2011/075595); Event 5307 (corn, insect control, deposited as ATCCPTA-9561, described in WO2010/077816); Event ASR-368 (bent grass,herbicide tolerance, deposited as ATCC PTA-4816, described inUS2006162007 or WO2004053062); Event B16 (corn, herbicide tolerance, notdeposited, described in US2003126634); Event BPS-CV127-9 (soybean,herbicide tolerance, deposited as NCIMB No. 41603, described inWO2010/080829); Event CE43-67B (cotton, insect control, deposited as DSMACC2724, described in US2009217423 or WO2006/128573); Event CE44-69D(cotton, insect control, not deposited, described in US20100024077);Event CE44-69D (cotton, insect control, not deposited, described inWO2006/128571); Event CE46-02A (cotton, insect control, not deposited,described in WO2006/128572); Event COT102 (cotton, insect control, notdeposited, described in US2006130175 or WO2004039986); Event COT202(cotton, insect control, not deposited, described in US2007067868 orWO2005054479); Event COT203 (cotton, insect control, not deposited,described in WO2005/054480); Event DAS40278 (corn, herbicide tolerance,deposited as ATCC PTA-10244, described in WO2011/022469); EventDAS-59122-7 (corn, insect control—herbicide tolerance, deposited as ATCCPTA 11384, described in US2006070139); Event DAS-59132 (corn, insectcontrol—herbicide tolerance, not deposited, described in WO2009/100188);Event DAS68416 (soybean, herbicide tolerance, deposited as ATCCPTA-10442, described in WO2011/066384 or WO2011/066360); EventDP-098140-6 (corn, herbicide tolerance, deposited as ATCC PTA-8296,described in US2009137395 or WO2008/112019); Event DP-305423-1 (soybean,quality trait, not deposited, described in US2008312082 orWO2008/054747); Event DP-32138-1 (corn, hybridization system, depositedas ATCC PTA-9158, described in US20090210970 or WO2009/103049); EventDP-356043-5 (soybean, herbicide tolerance, deposited as ATCC PTA-8287,described in US20100184079 or WO2008/002872); Event EE-1 (brinjal,insect control, not deposited, described in WO2007/091277); Event FI117(corn, herbicide tolerance, deposited as ATCC 209031, described inUS2006059581 or WO1998/044140); Event GA21 (corn, herbicide tolerance,deposited as ATCC 209033, described in US2005086719 or WO1998/044140);Event GG25 (corn, herbicide tolerance, deposited as ATCC 209032,described in US2005188434 or WO1998/044140); Event GHB119 (cotton,insect control—herbicide tolerance, deposited as ATCC PTA-8398,described in WO2008/151780); Event GHB614 (cotton, herbicide tolerance,deposited as ATCC PTA-6878, described in US2010050282 or WO2007/017186);Event GJ11 (corn, herbicide tolerance, deposited as ATCC 209030,described in US2005188434 or WO1998/044140); Event GM RZ13 (sugar beet,virus resistance, deposited as NCIMB-41601, described in WO2010/076212);Event H7-1 (sugar beet, herbicide tolerance, deposited as NCIMB 41158 orNCIMB 41159, described in US2004172669 or WO2004/074492); Event JOPLIN1(wheat, disease tolerance, not deposited, described in US2008064032);Event LL27 (soybean, herbicide tolerance, deposited as NCIMB41658,described in WO2006/108674 or US2008320616); Event LL55 (soybean,herbicide tolerance, deposited as NCIMB 41660, described inWO2006/108675 or US2008196127); Event LLcotton25 (cotton, herbicidetolerance, deposited as ATCC PTA-3343, described in WO2003013224 orUS2003097687); Event LLRICE06 (rice, herbicide tolerance, deposited asATCC-23352, described in U.S. Pat. No. 6,468,747 or WO2000/026345);Event LLRICE601 (rice, herbicide tolerance, deposited as ATCC PTA-2600,described in US20082289060 or WO2000/026356); Event LY038 (corn, qualitytrait, deposited as ATCC PTA-5623, described in US2007028322 orWO2005061720); Event MIR162 (corn, insect control, deposited asPTA-8166, described in US2009300784 or WO2007/142840); Event MIR604(corn, insect control, not deposited, described in US2008167456 orWO2005103301); Event MON15985 (cotton, insect control, deposited as ATCCPTA-2516, described in US2004-250317 or WO2002/100163); Event MON810(corn, insect control, not deposited, described in US2002102582); EventMON863 (corn, insect control, deposited as ATCC PTA-2605, described inWO2004/011601 or US2006095986); Event MON87427 (corn, pollinationcontrol, deposited as ATCC PTA-7899, described in WO2011/062904); EventMON87460 (corn, stress tolerance, deposited as ATCC PTA-8910, describedin WO2009/111263 or US20110138504); Event MON87701 (soybean, insectcontrol, deposited as ATCC PTA-8194, described in US2009130071 orWO2009/064652); Event MON87705 (soybean, quality trait—herbicidetolerance, deposited as ATCC PTA-9241, described in US20100080887 orWO2010/037016); Event MON87708 (soybean, herbicide tolerance, depositedas ATCC PTA9670, described in WO2011/034704); Event MON87754 (soybean,quality trait, deposited as ATCC PTA-9385, described in WO2010/024976);Event MON87769 (soybean, quality trait, deposited as ATCC PTA-8911,described in US20110067141 or WO2009/102873); Event MON88017 (corn,insect control—herbicide tolerance, deposited as ATCC PTA-5582,described in US2008028482 or WO2005/059103); Event MON88913 (cotton,herbicide tolerance, deposited as ATCC PTA-4854, described inWO2004/072235 or US2006059590); Event MON89034 (corn, insect control,deposited as ATCC PTA-7455, described in WO2007/140256 or US2008260932);Event MON89788 (soybean, herbicide tolerance, deposited as ATCCPTA-6708, described in US2006282915 or WO2006/130436); Event MS11(oilseed rape, pollination control—herbicide tolerance, deposited asATCC PTA-850 or PTA-2485, described in WO2001/031042); Event MS8(oilseed rape, pollination control—herbicide tolerance, deposited asATCC PTA-730, described in WO2001/041558 or US2003188347); Event NK603(corn, herbicide tolerance, deposited as ATCC PTA-2478, described inUS2007-292854); Event PE-7 (rice, insect control, not deposited,described in WO2008/114282); Event RF3 (oilseed rape, pollinationcontrol—herbicide tolerance, deposited as ATCC PTA-730, described inWO2001/041558 or US2003188347); Event RT73 (oilseed rape, herbicidetolerance, not deposited, described in WO2002/036831 or US2008070260);Event T227-1 (sugar beet, herbicide tolerance, not deposited, describedin WO2002/44407 or US2009265817); Event T25 (corn, herbicide tolerance,not deposited, described in US2001029014 or WO2001/051654); EventT304-40 (cotton, insect control—herbicide tolerance, deposited as ATCCPTA-8171, described in US2010077501 or WO2008/122406); Event T342-142(cotton, insect control, not deposited, described in WO2006/128568);Event TC1507 (corn, insect control—herbicide tolerance, not deposited,described in US2005039226 or WO2004/099447); Event VIP1034 (corn, insectcontrol—herbicide tolerance, deposited as ATCC PTA-3925., described inWO2003/052073), Event 32316 (corn, insect control-herbicide tolerance,deposited as PTA-11507, described in WO2011/084632) and Event 4114(corn, insect control-herbicide tolerance, deposited as PTA-11506,described in WO2011/084621).

TABLE 1 Overview of transgenic elite events and the nucleotide sequencesof the corresponding flanking sequences (all incorporated herein byreference). Plant Event name species Trait type Deposit Nr PatentReference Genomic flanking sequences 1143-14A COTTON INSECT CONTROL NONEWO 2006/128569 SEQ ID No 1: nt 1-316 SEQ ID No 2: nt 319-596 1143-51BCOTTON INSECT CONTROL NONE WO 2006/128570 SEQ ID No 1: nt 175-589 1445COTTON HERBICIDE TOLERANCE NONE WO 2002/034946 SEQ ID No 7: nt 1-172(5′) SEQ ID No 8: nt 375-499 (3′) 17053 RICE HERBICIDE TOLERANCE ATCCPTA-9843 WO 2010/117737 SEQ ID No 3: nt 1-574 (5′) SEQ ID No 4: nt 1-635(3′) 17314 RICE HERBICIDE TOLERANCE ATCC PTA-9844 WO 2010/117735 SEQ IDNo 3: nt 1-292 (5′) SEQ ID No 4: nt 1-665 (3′) 281-24-236 COTTON INSECTCONTROL - ATCC PTA-6233 WO 2005/103266 SEQ ID No 1: nt 1-2074 (5′)HERBICIDE TOLERANCE SEQ ID No 1: nt 12749-15490 (3′) 3006-210-23 COTTONINSECT CONTROL - ATCC PTA-6233 WO 2005/103266 SEQ ID No 2: nt 1-527 (5′)HERBICIDE TOLERANCE SEQ ID No 2: nt 8901-9382 (3′) 32316 CORN INSECTCONTROL - ATCC PTA-11507 WO 2011/084632 SEQ ID No 6: nt 1-2005 (5′)HERBICIDE TOLERANCE SEQ ID No 6: nt 13950-15992 (3′) 3272 CORN QUALITYTRAIT ATCC PTA-9972 WO 2006098952 SEQ ID No 5: (1409 nt) (′5) SEQ ID No6: (1557 nt) (3′) 40416 CORN INSECT CONTROL - ATCC PTA-11508 WO2011/075593 SEQ ID No 6: nt 1-508 (5′) HERBICIDE TOLERANCE SEQ ID No 6:nt 12369-13176 (3′) 4114 CORN INSECT CONTROL - ATCC PTA-11506 WO2011/084621 SEQ ID No 6: nt 1-2422 (5′) HERBICIDE TOLERANCE SEQ ID No 6:nt 14348-16752 (3′) 43A47 CORN INSECT CONTROL - ATCC PTA-11509 WO2011/075595 SEQ ID No 6: nt 1-987 (5′) HERBICIDE TOLERANCE SEQ ID No 6:nt 12899-14354 (3′) 5307 CORN INSECT CONTROL ATCC PTA-9561 WO2010/077816 SEQ ID No 5: (1548 nt) (5′) SEQ ID No 6: (1093 nt) (3′)ASR-368 BENT HERBICIDE TOLERANCE ATCC PTA-4816 WO 2004053062 SEQ ID No3: nt 1-637 (5′) GRASS SEQ ID No 4: nt 249-474 (3′) B16 CORN HERBICIDETOLERANCE NONE US 2003126634 — BPS- SOYBEAN HERBICIDE TOLERANCE NCIMBNo. 41603 WO 2010/080829 SEQ ID No 1: nt 1-1311 (5′) CV127-9 SEQ ID No1: nt 6070-10656 (3′) CE43-67B COTTON INSECT CONTROL DSM ACC2724 WO2006/128573 SEQ ID No 1: nt 1-275 (5′) SEQ ID No 2: 134-1198 (3′)CE44-69D COTTON INSECT CONTROL NONE WO 2006/128571 SEQ ID No 1: nt 1-135(5′) SEQ ID No 2: 272-659 (3′) CE46-02A COTTON INSECT CONTROL NONE WO2006/128572 SEQ ID No 1: nt 1-266 (5′) SEQ ID No 2: 150-530 (3′) COT102COTTON INSECT CONTROL NONE WO 2004/039986 SEQ ID No 5: (290 nt) (5′) SEQID No 6: (347 nt) (3′) COT202 COTTON INSECT CONTROL NONE WO 2005/054479SEQ ID No 7: (290 nt) (5′) SEQ ID No 8: (4382 nt) (3′) COT203 COTTONINSECT CONTROL NONE WO 2005/054480 SEQ ID No 7: (290 nt) (5′) SEQ ID No8: (4382 nt) (3′) DAS40278 CORN HERBICIDE TOLERANCE ATCC PTA-10244 WO2011/022469 SEQ ID No 29: nt 1-1873 (5′) SEQ ID No 29: nt 6690-8557 (3′)DAS- CORN INSECT CONTROL - ATCC PTA-11384 US 2006070139 SEQ ID No 19:(2593 nt) (5′) 59122-7 HERBICIDE TOLERANCE SEQ ID No 20: (1986 nt) (3′)DAS-59132 CORN INSECT CONTROL - NONE WO 2009/100188 SEQ ID No 3: nt118-870 (3′) HERBICIDE TOLERANCE DAS68416 SOYBEAN HERBICIDE TOLERANCEATCC PTA-10442 WO 2011/066384 SEQ ID No 1: nt 1-2730 (5′) SEQ ID No 1:nt 9122-10212 (3′) DAS68416 SOYBEAN HERBICIDE TOLERANCE ATCC PTA-10442WO 2011/066360 SEQ ID No 1: nt 1-2730 (5′) SEQ ID No 1: nt 9122-10212(3′) DP- CORN HERBICIDE TOLERANCE ATCC PTA-8296 WO 2008/112019 SEQ ID No48: (9423 nt) (5′ + insert + 3′) 098140-6 DP- SOYBEAN QUALITY TRAIT NONEUS 2008312082 SEQ ID NO: 5 nt 1-18651 (5′ contig1) 305423-1 SEQ ID NO: 5nt 31580-39,499 (3′ contig1) SEQ ID NO: 6 nt 1-12163 (5′ contig2) SEQ IDNO: 6 nt 14495-25843 (3′ contig3) SEQ ID NO: 7 nt 1-5750 (5′ contig3)SEQ ID NO: 7 nt 7814-12465 (3′ contig3) SEQ ID NO: 8 nt 1-2899 (5′contig4) SEQ ID NO: 8 nt 7910-10058 (3′ contig4) DP- CORN HYBRIDIZATIONSYSTEM ATCC PTA-9158 WO 2009/103049 SEQ ID NO: 5 nt 31580-39,499 (3′contig1) 32138-1 DP- SOYBEAN HERBICIDE TOLERANCE ATCC PTA-8287 WO2008/002872 SEQ ID NO: 8 nt 1-2899 (5′ contig4) 356043-5 EE-1 BRINJALINSECT CONTROL NONE WO 2007/091277 SEQ ID NO: 8 nt 7910-10058 (3′contig4) FI117 CORN HERBICIDE TOLERANCE ATCC 209031 WO 1998/044140 —GA21 CORN HERBICIDE TOLERANCE ATCC 209033 WO 1998/044140 — GG25 CORNHERBICIDE TOLERANCE ATCC 209032 WO 1998/044140 — GHB119 COTTON INSECTCONTROL - ATCC PTA-8398 WO 2008/151780 SEQ ID No 11: nt 1-463 (5′)HERBICIDE TOLERANCE SEQ ID No 2: nt 1-112 (3′) GHB614 COTTON HERBICIDETOLERANCE ATCC PTA-6878 WO 2007/017186 SEQ ID No 1: nt 1-732 (5′) SEQ IDNo 2: nt 1-430 (3′) GJ11 CORN HERBICIDE TOLERANCE ATCC 209030 WO1998/044140 — GM RZ13 SUGAR VIRUS RESISTANCE NCIMB-41601 WO 2010/076212SEQ ID No 9: (237 nt) (5′) BEET SEQ ID No 3: (347 nt) (3′) H7-1 SUGARHERBICIDE TOLERANCE NCIMB 41158 or WO 2004/074492 SEQ ID No 5: (3778 nt)(5′ + insert + 3′) BEET NCIMB 41159 JOPLIN1 WHEAT DISEASE TOLERANCE NONEUS 2008064032 SEQ ID No 1: nt 1-1393 (5′) SEQ ID No 2: nt 427-2471 (3′)LL27 SOYBEAN HERBICIDE TOLERANCE NCIMB41658 WO 2006/108674 SEQ ID No 1:nt 1-209 (5′) SEQ ID No 2: nt 569-1000 (3′) LL55 SOYBEAN HERBICIDETOLERANCE NCIMB 41660 WO 2006/108675 SEQ ID No 1: nt 1-311 (5′) SEQ IDNo 2: nt 510-1880 (3′) LLcotton25 COTTON HERBICIDE TOLERANCE ATCCPTA-3343 WO 2003/013224 SEQ ID No 1: nt 1-677 (5′) SEQ ID No 2: nt180-426 (3′) LLRICE06 RICE HERBICIDE TOLERANCE ATCC-23352 WO 2000/026345SEQ ID No 1: nt 1-92 (5′) SEQ ID No 2: nt 605-1279 (3′) LLRICE601 RICEHERBICIDE TOLERANCE ATCC PTA-2600 US 2008289060 SEQ ID No 1: nt 1-603(5′) SEQ ID No 2: nt 73-607 (3′) LLRICE62 RICE HERBICIDE TOLERANCEATCC-203353 WO 2000/026356 SEQ ID No 1: nt 604-1009 (3′) LY038 CORNQUALITY TRAIT ATCC PTA-5623 WO 2005/061720 SEQ ID No 1: nt 1-1781 (5′proximal) SEQ ID No 9: (1736 nt) (5′ distal) SEQ ID No 2: nt 201-867 (3′proximal) SEQ ID No 10: (359 nt) (3′ distal) MIR162 CORN INSECT CONTROLATCC PTA-8166 WO 2007/142840 SEQ ID No 46: (1088 nt) (5′) SEQ ID No 48:(1189 nt) (3′) MIR604 CORN INSECT CONTROL NONE WO 2005/103301 SEQ ID No5: (801 nt) (5′) SEQ ID No 6: (1064 nt) (3′) MON15985 COTTON INSECTCONTROL ATCC PTA-2516 WO 2002/100163 SEQ ID No 4: 531 (5′ MON531) SEQ IDNo 6: (3′ MON531) SEQ ID No 19: (5′ MON15985) SEQ ID No 25: (3′MON15985) MON810 CORN INSECT CONTROL NONE US 2002102582 SEQ ID No 5:(244 nt) (5′) SEQ ID No 6: (606 nt) (3′) MON863 CORN INSECT CONTROL ATCCPTA-2605 WO 2004/011601 SEQ ID No 5: (242 nt) (5′) SEQ ID No 6: (224 nt)(3′) MON87427 CORN POLLINATION CONTROL ATCC PTA-7899 WO 2011/062904 SEQID No 7: (972 nt) (5′ + part of transgene) SEQ ID No 8: (1078 nt) (3′ +part of transgene) MON87460 CORN STRESS TOLERANCE ATCC PTA-8910 WO2009/111263 SEQ ID No 5: (1060 nt) (5′) SEQ ID No 6: (1260 nt) (3′)MON87701 SOYBEAN INSECT CONTROL ATCC PTA-8194 WO 2009/064652 SEQ ID No3: nt 1-5747 (5′) SEQ ID No 4: nt 289-2611 (3′) MON87705 SOYBEAN QUALITYTRAIT - ATCC PTA-9241 WO 2010/037016 SEQ ID No 3: (3458 nt) (5′)HERBICIDE TOLERANCE SEQ ID No 4: (2515 nt) (3′) MON87708 SOYBEANHERBICIDE TOLERANCE ATCC PTA9670 WO 2011/034704 SEQ ID No 3: nt 1-1126(5′) SEQ ID No 4: nt 131-1947 (3′) MON87754 SOYBEAN QUALITY TRAIT ATCCPTA-9385 WO 2010/024976 SEQ ID No 3: nt 1-942 (5′) SEQ ID No 4: nt154-1244 (3′) MON87769 SOYBEAN QUALITY TRAIT ATCC PTA-8911 WO2009/102873 SEQ ID No 3: nt 1-978 (5′) SEQ ID No 4: nt 10-939 (3′)MON88017 CORN INSECT CONTROL - ATCC PTA-5582 WO 2005/059103 SEQ ID No 3:(1461 nt) HERBICIDE TOLERANCE (5′ + part of transgene) SEQ ID No 4:(3525 nt) (3′ + part of transgene) MON88913 COTTON HERBICIDE TOLERANCEATCC PTA-4854 WO 2004/072235 SEQ ID No 3: (2880 nt) (5′ + part oftransgene) SEQ ID No 4: (1675) (3′ + part of transgene) MON89034 CORNINSECT CONTROL ATCC PTA-7455 WO 2007/140256 SEQ ID No 3: nt 1-2050 (5′)SEQ ID No 4: nt 1-914 (3′) MON89788 SOYBEAN HERBICIDE TOLERANCE ATCCPTA-6708 WO 2006/130436 SEQ ID No 3: (1222 nt) (5′ + part of T-DNA) SEQID No 4: (1675 nt) (3′ + part of T-DNA) MS11 OILSEED POLLINATIONCONTROL - ATCC PTA-850 or WO 2001/031042 SEQ ID No 8: nt 1-234 (5′) RAPEHERBICIDE TOLERANCE PTA-2485 SEQ ID No 10: nt 194-416 (3′) MS8 OILSEEDPOLLINATION CONTROL - ATCC PTA-730 WO 2001/041558 SEQ ID No 13: 1-867(5′) RAPE HERBICIDE TOLERANCE SEQ ID No 18: nt 181-537 (3′) NK603 CORNHERBICIDE TOLERANCE ATCC PTA-2478 US 2004-139493 SEQ ID No 7: nt 1-304(5′) SEQ ID No 8: nt 687-1183 (3′) PE-7 RICE INSECT CONTROL NONE WO2008/114282 SEQ ID No 7: nt 31-875 (left border) RF3 OILSEED POLLINATIONCONTROL - ATCC PTA-730 WO 2001/041558 SEQ ID No 24: nt 1-881 (5′) RAPEHERBICIDE TOLERANCE SEQ ID No 30: nt 167-1441 (3′) RT73 OILSEEDHERBICIDE TOLERANCE NONE WO 2002/036831 SEQ ID No 7: (5′ + part ofT-DNA) RAPE SEQ ID No 8: (3′ + part of T-DNA T227-1 SUGAR HERBICIDETOLERANCE NONE WO 2002/044407 FIG. 4.: (right bonder) BEET FIG. 7: (leftborder) T25 CORN HERBICIDE TOLERANCE NONE WO 2001/051654 SEQ ID No 6: nt1-341 (5′) SEQ ID No 10: nt 343-484 (3′) T304-40 COTTON INSECT CONTROL -ATCC PTA-8171 WO 2008/122406 SEQ ID No 16: nt 1-563 (5′) HERBICIDETOLERANCE SEQ ID No 2: nt 41-452 (3′) T342-142 COTTON INSECT CONTROLNONE WO 2006/128568 SEQ ID No 1: nt 1-162 (5′) SEQ ID No 2: nt 265-570(3′) TC1507 CORN INSECT CONTROL - NONE WO 2004/099447 SEQ ID No 21:(2829 nt) (5′) HERBICIDE TOLERANCE SEQ ID No 22: (2346 nt) (3′) VIP1034CORN INSECT CONTROL - ATCC PTA-3925. WO 2003/052073 SEQ ID No 11: (716nt) (5′ vip3A) HERBICIDE TOLERANCE SEQ ID No 12: (768 nt) (3′ vip3A) SEQID No 15: (94 nt) (5′ fragmented vip3A) SEQ ID No 13: (754 nt) (5′ pat)SEQ ID No 14: (94 nt) (3′ pat)

Nt 7060-7976: Ph4a748: sequence including the promoter region of thehistone H4 gene of Arabidopsis thaliana (Chaboute et al., 1987). As usedherein “in close proximity”, refers to the predefined site being locatedat such a distance from the existing transgenic event so as that theintroduced modification in the vicinity of or at the predefined sitewill be genetically linked to the existing event, i.e. they will inheritas a single genetic unit in at least 99% of the cases. Genetic linkageis usually expressed in terms of centimorgans (abbreviated cM).Centimorgan is a unit of recombinant frequency for measuring geneticlinkage, defined as that distance between genes for which one product ofmeiosis in 100 is recombinant, or in other words, the centimorgan isequal to a 1% chance that a marker at one genetic locus on a chromosomewill be separated from a marker at a second locus due to crossing overin a single generation. It is often used to infer distance along achromosome. The number of base-pairs to which cM correspond varieswidely across the genome (different regions of a chromosome havedifferent propensities towards crossover) and the species (i.e. thetotal size of the genome). For instance, the tetraploid cotton genomehas been estimated to include about 2200-3000 Mb of DNA distributedacross 26 chromosomes, with a total recombinational length of about 400kb per centimorgan (Smith and Cothren, “Cotton: origin, history,technology and production”, p 421). In A. thaliana, 1 cM corresponds toapproximately 217 kb, while in e.g. Z. mays this is about 1460 kb(Mézard C. Meiotic recombination hotspots in plants. Biochem Soc Trans.2006 August 34:531-4; Civardi et al., The relationship between geneticand physical distances in the cloned a1-sh2 interval of the Zea mays L.genome. Proc Natl Acad Sci USA. 1994 91(17):8268-72, p. 8271; fromhttp://bionumbers.hms.harvard.edu/default.aspx). “In close proximity”,as used herein, thus refers to at least a 99% chance that themodification and the elite event will inherit as a single genetic unitfor at least one generation, and therefore means within 1 cM, within 0.5cM, within 0.1 cM, within 0.05 cM, within 0.01 cM, within 0.005 cM orwithin 0.001 cM of the elite event. Relating to base pairs, “in closeproximity” can refer to within 5000 kb, within 1000 kb, within 500 kb,within 100 kb, within 50 kb, within 10 kb, within 5 kb, within 4 kb,within 3 kb, within 2 kb, within 1 kb, within 750 bp, or within 500 bpfrom the existing elite event (depending on the species and location inthe genome), e.g. between 1 kb and 10 kb or between 1 kb and 5 kb fromthe existing elite event.

It will be clear that the predefined site as well as the recognitionsite should be located such as not to interfere in a negative mannerwith the existing elite event. Vice versa, the existing elite eventshould also not negatively influence function of the newly introducedmodification, e.g. the functional expression of the newly insertedtransgene. It is presently demonstrated that it is possible to make amodification in close proximity to an existing elite event, for examplein one of the flanking sequences of the event, which does not negativelyaffect the existing event (in this case glufosinate tolerance orLepidoptera resistance) and results in good functional expression of thenew modification (e.g. glyphosate tolerance or HPPD inhibitor herbicidetolerance). In one embodiment, “in close proximity” may therefore referto within one of the flanking sequences of the elite event.

A preferred event in the context of this invention is cotton eventGHB119, also known as EE-GH6 (described in 2008/151780, there alsoreferred to as GBH119, deposit nr ATCC PTA-8398). SEQ ID NO. 4represents the nucleotide sequence of the 5′ flanking sequence of theGHB119 event and SEQ ID No. 3 represents the nucleotide sequence of the3′ flanking sequence of the GHB119 event, the latter of which contains arecognition site for the herein described COT-5/6 meganuclease (SEQ IDNO. 1 and its reverse complement SEQ ID NO. 2). The recognition site ofSEQ ID No. 1 corresponds to the nucleotide sequence of SEQ ID No. 3 fromnucleotide 2114 to 2135. The herein described meganucleases are thuscapable of recognizing and cleaving a nucleotide sequence in closeproximity of an existing transgenic event, such as GHB119.

Thus, in one embodiment, the predefined site and/or recognition siteis/are located in one of the flanking sequences of the elite event.Flanking sequences of elite events which are encompassed in theinvention are listed non-exhaustively in table 1. The nucleotidesequences of the flanking sequences of elite event GHB119 arerepresented by SEQ ID NO: 3 (3′) and SEQ ID NO: 4 (5′).

In another embodiment, the recognition sequence comprises the nucleotidesequence of SEQ ID NO: 1 or 2.

The redesigned meganucleases described herein are based on the naturallyoccurring meganuclease I-CreI for use as a scaffold. I-CreI is a homingendonuclease found in the chloroplasts of Chlamydomonas rheinhardti(Thompson et al. 1992, Gene 119, 247-251). This endonuclease is ahomodimer that recognizes a pseudo-palindromic 22 by DNA site in the23SrRNA gene and creates a double stranded DNA break that is used forthe introduction of an intron. I-CreI is a member of a group ofendonucleases carrying a single LAGLIDADG motif. LAGLIDADG enzymescontain one or two copies of the consensus motif. Single-motif enzymes,such as I-CreI function as homodimers, whereas double-motif enzymes aremonomers with two separate domains. Accordingly, when re-designingmeganucleases derived from an I-CreI scaffold to recognize a 22 bpnucleotide sequence of interest, two monomeric units are designed, eachrecognizing a part of the 22 bp recognition site, which are needed inconcert to induce a double stranded break at the 22 bp recognition site(WO2007/047859). Concerted action may be achieved by linking the twomonomeric units into one single chain meganuclease, or may also beachieved by promoting the formation of heterodimers, as described e.g.in WO2007/047859. Examples of such specifically designed meganucleasesare described in e.g. EP10005926.0 and EP10005941.9 (unpublished).

The amino acid sequence of a naturally occurring I-CreI monomer isprovided as SEQ ID No. 16. To re-design I-CreI monomeric units such thatthe heterodimers thereof recognize the nucleotide sequence of SEQ ID No.1 and/or 2, the following amino acids are present at the mentionedpositions in meganuclease unit 1:

-   -   a. S at position 32;    -   b. Y at position 33;    -   c. Q at position 38;    -   d. Q at position 80;    -   e. S at position 40;    -   f. T at position 42;    -   g. R at position 77;    -   h. Y at position 68;    -   i. Q at position 70;    -   j. H at position 75;    -   k. T at position 44;    -   l. I at position 24;    -   m. Q at position 26;    -   n. K at position 28;    -   o. N at position 30.

and wherein the following amino acids are present in meganuclease unit2:

-   -   p. S at position 70;    -   q. Q at position 44;    -   r. K at position 24;    -   s. A at position 26;    -   t. K at position 28;    -   u. N at position 30;    -   v. S at position 32;    -   w. Y at position 33;    -   x. Q at position 38;    -   y. Q at position 80;    -   z. S at position 40;    -   aa. T at position 42;    -   bb. Q at position 77;    -   cc. Y at position 68.        A schematic representation thereof is provided in FIG. 1.        Optionally, a glutamine (Gln/Q) may be present in both subunits        at position 47 with respect to the I-CreI amino acid sequence.

The re-designed double stranded break inducing enzyme may comprise, butneed not comprise, a nuclear localization signal (NLS), such as the NLSof SV40 large T-antigen [Raikhel, Plant Physiol. 100: 1627-1632 (1992)and references therein] [Kalderon et al. Cell 39: 499-509 (1984)]. Thenuclear localization signal may be located anywhere in the protein, butis conveniently located at the N-terminal end of the protein. Thenuclear localization signal may replace one or more of the amino acidsof the double stranded break inducing enzyme. It should be noted that ifthe re-designed meganuclease has been provided with a NLS at theN-terminus of the protein, such as a 10 or 12 amino acid NLS of SV40,the amino acid positions would be shifted (increased) accordingly.Likewise, in the event two monomeric units are linked into a singlechain meganuclease, the position of the second unit will also beshifted. The corresponding amino acid positions with regard to theI-CreI amino acid sequence (SEQ ID NO. 16) can also be identified bydetermining the optimal alignment as described below. It will be clearthat in the single chain redesigned meganuclease the order of the unitsis irrelevant, i.e. whether the above unit 1 and 2 occur indeed withinthat order in the single amino acid chain or unit 2 precedes unit one inthe single amino acid chain does not make a difference in order for thetwo units combined to be able to recognize the target sequence.

Re-designed meganucleases suitable for the invention may comprise anamino acid sequence as represented by SEQ ID No. 6, which encodes asingle chain meganuclease comprising two subunits (amino acids 11-165and 204-360, respectively) coupled by a linker sequence (amino acids166-203) and preceded by a nuclear localization sequence (amino acids1-10). Alternatively such meganucleases may consist of two monomericunits which can cleave the recognition site as a heterodimer. Such aheterodimeric meganuclease may also comprise the amino acids sequencesof SEQ ID NO 5. from amino acid position 11-165 and 204-360.

Conveniently, the DSBI enzyme can be provided by expression of a plantexpressible recombinant (chimeric) gene(s) encoding suchmeganuclease(s). To this end, a DNA region comprising a nucleotidesequence encoding a re-designed meganuclease or meganuclease monomericunit can be operably linked to a plant-expressible promoter andoptionally a DNA region involved in transcription termination andpolyadenylation and introduced into a plant, plant part or plant cells.The recombinant gene(s) encoding DSBI enzyme may be introducedtransiently or stably. The DSBI enzyme may also be introduced into theplant, plant part or plant cell by introducing into the cell an RNAmolecule which is translated into the DSBI enzyme. Alternatively, theDSBI enzyme may be introduced into the plant, plant part or plant celldirectly as a protein. Methods for the introduction of DNA or RNAmolecules or proteins into a plant, plant part, tissue or plant cell aredescribed elsewhere in this application.

For the purpose of the invention, the term “plant-operative promoter”and “plant-expressible promoter” mean a promoter which is capable ofdriving transcription in a plant, plant tissue, plant organ, plant part,or plant cell. This includes any promoter of plant origin, but also anypromoter of non-plant origin which is capable of directing transcriptionin a plant cell.

Promoters that may be used in this respect are constitutive promoters,such as the promoter of the cauliflower mosaic virus (CaMV) 35Stranscript (Hapster et al., 1988, Md. Gen. Genet. 212: 182-190), theCaMV 19S promoter (U.S. Pat. No. 5,352,605; WO 84/02913; Benfey et al.,1989, EMBO J. 8:2195-2202), the subterranean clover virus promoter No 4or No 7 (WO 96/06932), the Rubisco small subunit promoter (U.S. Pat. No.4,962,028), the ubiquitin promoter (Holtorf et al., 1995, Plant Mol.Biol. 29:637-649), T-DNA gene promoters such as the octopine synthase(OCS) and nopaline synthase (NOS) promoters from Agrobacterium, andfurther promoters of genes whose constitutive expression in plants isknown to the person skilled in the art.

Further promoters that may be used in this respect are tissue-specificor organ-specific promoters, preferably seed-specific promoters, such asthe 2S albumin promoter (Joseffson et al., 1987, J. Biol. Chem.262:12196-12201), the phaseolin promoter (U.S. Pat. No. 5,504,200;Bustos et al., 1989, Plant Cell 1. (9):839-53), the legumine promoter(Shirsat et al., 1989, Mol. Gen. Genet. 215(2):326-331), the “unknownseed protein” (USP) promoter (Baumlein et al., 1991, Mol. Gen. Genet.225(3):459-67), the napin promoter (U.S. Pat. No. 5,608,152; Stalberg etal., 1996, Planta 199:515-519), the Arabidopsis oleosin promoter (WO98/45461), the Brassica Bce4 promoter (WO 91/13980), and furtherpromoters of genes whose seed-specific expression in plants is known tothe person skilled in the art.

Other promoters that can be used are tissue-specific or organ-specificpromoters like organ primordia-specific promoters (An et al., 1996,Plant Cell 8: 15-30), stem-specific promoters (Keller et al., 1988, EMBOJ. 7(12): 3625-3633), leaf-specific promoters (Hudspeth et al., 1989,Plant Mol. Biol. 12: 579-589), mesophyl-specific promoters (such as thelight-inducible Rubisco promoters), root-specific promoters (Keller etal., 1989, Genes Dev. 3: 1639-1646), tuber-specific promoters (Keil etal., 1989, EMBO J. 8(5): 1323-1330), vascular tissue-specific promoters(Peleman et al., 1989, Gene 84: 359-369), stamen-selective promoters (WO89/10396, WO 92/13956), dehiscence zone-specific promoters (WO97/13865), and the like.

Nucleotide sequences encoding DSBI enzymes (re-designed meganucleases)suitable for the invention may comprise the nucleotide sequence of SEQID No. 5 from nucleotide position 3120-3584 and the nucleotide sequenceof SEQ ID No. 5 from nucleotide position 3698-4169 (excluding linker andNLS). In case of a single chain meganuclease, this may include a linkersequence between the two subunit, such as a linker sequence as encodedby the nucleotide sequence of SEQ ID NO. 5 from nucleotide position3584-3697. A nucleotide sequence encoding a nuclear localization signalmay also be included in the DSBI enzyme encoding nucleotide sequences,such as the nucleotide sequence of SEQ ID NO. 5 from position 3091-3119.To facilitate cloning and other recombinant DNA techniques, it may beadvantageous to include an intron functional in plants into the regionencoding a meganuclease, particularly a single chain meganuclease.

The DNA region encoding the DSBI enzyme may be optimized for expressionin plants by adapting GC content, codon usage, elimination of unwantednucleotide sequences. The coding region may further be optimized forexpression in plants and the synthetic coding region may have anucleotide sequence which has been designed to fulfill the followingcriteria:

-   -   a) the nucleotide sequence encodes a functional redesigned        homing endonuclease as herein described;    -   b) the codon usage is adapted to the preferred codon usage of        the target organism;    -   c) the nucleotide sequence has a GC content of about 50% to        about 60%;    -   d) the nucleotide sequence does not comprise a nucleotide        sequence selected from the group consisting of GATAAT, TATAAA,        AATATA, AATATT, GATAAA, AATGAA, AATAAG, AATAAA, AATAAT, AACCAA,        ATATAA, AATCAA, ATACTA, ATAAAA, ATGAAA, AAGCAT, ATTAAT, ATACAT,        AAAATA, ATTAAA, AATTAA, AATACA and CATAAA;    -   e) the nucleotide does not comprise a nucleotide sequence        selected from the group consisting of CCAAT, ATTGG, GCAAT and        ATTGC;    -   f) the nucleotide sequence does not comprise a sequence selected        from the group consisting of ATTTA, AAGGT, AGGTA, GGTA or GCAGG;    -   g) the nucleotide sequence does not comprise a GC stretch        consisting of 7 consecutive nucleotides selected from the group        of G or C;    -   h) the nucleotide sequence does not comprise a AT stretch        consisting of 5 consecutive nucleotides selected from the group        of A or T; and    -   i) the nucleotide sequence does not comprise codons coding for        Leu, Ile, Val, Ser, Pro, Thr, Ala that comprise TA or CG duplets        in positions 2 and 3 (i.e. the nucleotide sequence does not        comprise the codons TTA, CTA, ATA, GTA, TCG, CCG, ACG and GCG).

It will also be clear that the terms used to describe the method such as“introduction of a DNA fragment” as well as “regeneration of a plantfrom the cell” do not imply that such DNA fragment necessarily needs tobe introduced by transformation techniques. Indeed, it will beimmediately clear to the person skilled in the art that the DNA moleculeof interest may also be introduced by breeding or crossing techniquesfrom one plant to another. Thus, “Introducing” in connection with thepresent application relate to the placing of genetic information in aplant cell or plant by any known means. This can be effected by anymethod known in the art for transforming RNA or DNA into plant cells,tissues, protoplasts or whole plants or by introgressing said RNA or DNAinto plants as described below. More particularly, “introducing” meansstably integrating into the plant's genome.

Nucleic acid molecules may be introduced into the plant cells by anymethod known in the art, including Agrobacterium-mediated transformationbut also by direct DNA transfer methods. Various methods for DNAdelivery into cells/tissues (intact plant cells or partially degradedtissues or plant cells) are known in the art, and includeelectroporation as illustrated in U.S. Pat. No. 5,384,253;microprojectile bombardment (biolistics) as illustrated in U.S. Pat.Nos. 5,015,580; 5,550,318; 5,538,880; 6,160,208; 6,399,861; and6,403,865; Cotton transformation by particle bombardment is reportede.g. in WO 92/15675; Agrobacterium-mediated transformation asillustrated in U.S. Pat. Nos. 5,635,055; 5,824,877; 5,591,616;5,981,840; and 6,384,301; Agrobacterium-mediated transformation ofcotton has been described e.g. in U.S. Pat. No. 5,004,863, in U.S. Pat.No. 6,483,013 and WO2000/71733.; protoplast transformation asillustrated in U.S. Pat. No. 5,508,184, electroporation,chemically-assisted transformation, liposome-mediated transformation(see, e.g., A. Deshayes, et al. (1985) EMBO J. 4:2731-7.), carbon fiber,silicon carbide fiber or aluminum borate fiber (generally termedwhiskers) (see, e.g., J. Brisibe, Exp. Bot. 51 (343):187-196 (2000);Dunwell (1999) Methods Mol. Biol. 1 11:375-82; and U.S. Pat. No.5,464,765), micro-injection (see, e.g., TJ. Reich, of al. (1986)Biotechnology 4: 1001-1004) and viral-mediated transformation (see,e.g., S. B. Gelvin, (2005) Nat Biotechnol. 23:684-5, WO 90/12107, WO03/052108 and WO 2005/098004), bombardment of plant cells withheterologous foreign DNA adhered to particles, ballistic particleacceleration, aerosol beam transformation (U.S. Patent Application No.20010026941; U.S. Pat. No. 4,945,050; International Publication No. WO91/00915; U.S. Patent Application No. 2002015066, WO 01/038514; allincorporated herein by reference), Led transformation, PEGtransformation, and various other non-particle direct-mediated methodsto transfer DNA. As used herein “direct DNA transfer” is any method ofDNA introduction into plant cells which does not involve the use ofnatural Agrobacterium spp. and which is capable of introducing DNA intoplant cells.

In one embodiment, the nucleic acid molecule(s), such as the repair DNAand/or the DSBI enzyme expression construct, is/are introduced into theplant cell by direct DNA transfer, e.g via particle bombardment. Inanother embodiment, introduction of DNA molecules takes place usingAgrobacterium.

In a specific embodiment, the plant cells are comprised withinembryogenic callus, preferable friable callus (i.e. the plant cells arecallus cells). The term “callus” or “embryogenic callus” refers to adisorganized mass of mainly embryogenic cells and cell clusters producedas a consequence of plant tissue culture. Friable callus refers tocallus with a friable texture with the potential to form shoots androots and eventually regenerate into whole plants. Such callus canfurther be distinguished by a parrot-green/creamy color, readilydispersed cell clumps in liquid medium, and a nodular shape. Callus canbe regenerated/induced from various tissue explants, such as hypocotyl,cotyledon, immature zygotic embryos, leaves, anthers, microspores,petals, ovules, roots, and meristems, stem cells and petioles.Transformation of embryogenic callus for the purpose of targeted genomemodification in cotton plants cells is described in U.S. provisionalapplication 61/493,579 and EP11004570.5 (herein incorporated byreference, in particular pages 12-15, paragraphs 40-50, and pages 31-35,examples 2-5).

The capability of inducing a double stranded break at a preselected siteopens up several potential applications, i.e. insertion, replacement ordeletion of one or more nucleotides. In case a DNA of interest presentin the repair DNA molecule is to be inserted into the preselected site,this can occur by either homologous recombination, or by the process ofnon-homologous end-joining. The double stranded break may also be usedto induce the formation of small deletions or insertions at thepreselected site, thereby potentially inactivating a gene or regulatoryelement comprising the nucleotide sequence of the preselected site. Thedouble stranded break at the preselected site will also facilitatereplacement of a DNA region in the vicinity of that site for a DNA ofinterest using a repair DNA, e.g. as described in WO 06/105946,WO08/037436 or WO08/148559.

If the double stranded DNA break induction is accompanied by theintroduction of a repair DNA molecule which is used as a template, thedouble stranded break repair can occur basically in three ways. Therepair DNA can be integrated into the genomic DNA at the DSB site bynon-homologous end joining at both ends, or if one or two flankingregions with homology to the up- and/or downstream regions of thepreselected site (the homology regions) are present in the repair DNA,integration of the repair DNA can also occur (partly) through homologousrecombination. As such, the double stranded break at the preselectedsite will also facilitate replacement of a DNA region in the vicinity ofthat site for a DNA region of interest e.g. as described in WO06/105946, WO08/037436 or WO08/148559.

To insert a DNA of interest by homologous recombination at thepreselected site, the repair DNA may comprise at least one flanking DNAregion having a nucleotide sequence which is similar to the nucleotidesequence of the DNA region upstream or downstream of the preselectedsite. The foreign DNA may also comprise two flanking DNA regions,located on opposite ends of the molecule and which have sufficienthomology to nucleotide sequence of the DNA region upstream anddownstream of the preselected site respectively to allow recombinationbetween said flanking regions and said upstream and downstream region.

As used herein “a flanking DNA region” is a DNA region in the repair DNAwith a nucleotide sequence having homology (i.e. high sequence identity)to the DNA regions respectively upstream or downstream of the target DNAsequence or preselected site (the homology regions). This allows tobetter control the insertion of DNA of interest. Indeed, integration byhomologous recombination will allow precise joining of the DNA ofinterest to the plant nuclear genome up to the nucleotide level.

To have sufficient homology for recombination, the flanking DNA regionsof the repair DNA may vary in length, and should be at least about 10nucleotides in length. However, the flanking region may be as long as ispractically possible (e.g. up to about 100-150 kb such as completebacterial artificial chromosomes (BACs). Preferably, the flanking regionwill be about 50 by to about 2000 bp. Moreover, the regions flanking theDNA of interest need not be identical to the homology regions (the DNAregions flanking the preselected site) and may have between about 80% toabout 100% sequence identity, preferably about 95% to about 100%sequence identity with the DNA regions flanking the preselected site.The longer the flanking region, the less stringent the requirement forhomology. Furthermore, it is preferred that the sequence identity is ashigh as practically possible in the vicinity of the DSB. Furthermore, toachieve exchange of the target DNA sequence without changing the DNAsequence of the adjacent DNA sequences, the flanking DNA sequencesshould preferably be identical to the upstream and downstream DNAregions flanking the preselected site or the target DNA sequence to beexchanged. The same criteria apply for recombination between theupstream and downstream region bearing homology to each other to removethe intervening DNA sequences by intrachromosomal homologousrecombination.

Moreover, the flanking region(s) of the repair DNA do not need to havehomology to the regions immediately flanking the preselected site, butmay have homology to a DNA region of the nuclear genome further remotefrom that preselected site. Insertion of the DNA of interest will thenresult in a removal of the target DNA between the preselected insertionsite and the DNA region of homology. In other words, the target DNAlocated between the homology regions (i.e. the genomic regions withhomology to the flanking regions of the foreign repair DNA) will besubstituted for the DNA of interest located between the two flankingregions of the repair DNA. When the repair DNA consists of the twoflanking regions only, i.e. lacking any intervening sequences (DNA ofinterest), this approach can be used to specifically delete the genomicregion located between the two homology regions.

As used herein “a preselected site” or “predefined site” indicates aparticular nucleotide sequence in the plant genome (e.g. the nucleargenome) located in or near the target DNA sequence at which location itis desired to insert, replace or delete one or more nucleotides. Aperson skilled in the art would be able to either choose a doublestranded DNA break inducing (“DSBI”) enzyme recognizing the selectedtarget nucleotide sequence or engineer such a DSBI endonuclease.Alternatively, a DSBI enzyme recognition site may be introduced into theplant genome using any conventional transformation method or byconventional breeding using a plant line having a DSBI endonucleaserecognition site in its genome, and any desired DNA may afterwards beintroduced into that preselected site.

In a further embodiment, the invention provides the use of a DSBIenzyme, such as a non-naturally occurring DSBI enzyme as describedabove, to modify the genome of a plant cell in the proximity of anexisting elite event.

As used herein “located in the vicinity” refers to the site of doubleDNA stranded break induction, i.e. the recognition site of the DSBIenzyme, being located at a distance of 100 bp, 250 bp, 500 bp, 1 kbp, 2kbp, 3 kbp, 4 kbp, 5 kbp to 10 kbp from the predefined site, i.e. thesite in the genomic DNA which is to be modified (the target site).

The DNA of interest to be inserted may also comprise a selectable orscreenable marker, which may or may not be removed after insertion.

“Selectable or screenable markers” as used herein have there usualmeaning in the art and include, but are not limited to plant expressiblephosphinotricin acetyltransferase, neomycine phosphotransferase,glyphosate oxidase, glyphosate tolerant EPSP enzyme, nitrilase gene,mutant acetolactate synthase or acetohydroxyacid synthase gene,β-glucoronidase (GUS), R-locus genes, green fluorescent protein and thelikes.

The selection of the plant cell or plant wherein the selectable orscreenable marker and the rest of the DNA of interest has beenintroduced by homologous recombination through the flanking DNA regionscan e.g. be achieved by screening for the absence of sequences presentin the transforming DNA but located outside of the flanking DNA regions.Indeed, presence of sequences from the transforming DNA outside theflanking DNA regions would indicate that the origination of thetransformed plant cells is by random DNA insertion. To this end,selectable or screenable markers may be included in the transforming DNAmolecule outside of the flanking DNA regions, which can then be used toidentify those plant cells which do not have the selectable orscreenable markers located outside of the transforming DNA and which mayhave arisen by homologous recombination through the flanking DNAregions. Alternatively, the transforming DNA molecule may containselectable markers outside the flanking DNA regions that allow selectionfor the absence of such genes (negative selectable marker genes).

It will be clear that the methods according to the invention allowinsertion of any DNA of interest including DNA comprising a nucleotidesequence with a particular nucleotide sequence signature e.g. forsubsequent identification, or DNA comprising (inducible) enhancers orsilencers, e.g. to modulate the expression of the existing elite event.The DNA of interest may also comprise one or more plant expressiblegene(s) of interest including but not limited to a herbicide tolerancegene, an insect resistance gene, a disease resistance gene, an abioticstress resistance gene, an enzyme involved in oil biosynthesis orcarbohydrate biosynthesis, an enzyme involved in fiber strength and/orlength, an enzyme involved in the biosynthesis of secondary metabolites.Particular mention may be made of herbicide-tolerance genes conferingtolerance to the herbicides inhibiting the enzymehydroxyphenylpyruvatedioxygenase (HPPD).Hydroxyphenylpyruvatedioxygenases are enzymes that catalyze the reactionin which para-hydroxyphenylpyruvate (HPP) is transformed intohomogentisate. Plants tolerant to HPPD-inhibitors can be transformedwith a gene encoding a naturally-occurring resistant HPPD enzyme, or agene encoding a mutated or chimeric HPPD enzyme as described in WO96/38567, WO 99/24585, and WO 99/24586, WO 2009/144079, WO 2002/046387,or U.S. Pat. No. 6,768,044. Tolerance to HPPD-inhibitors can also beobtained by transforming plants with genes encoding certain enzymesenabling the formation of homogentisate despite the inhibition of thenative HPPD enzyme by the HPPD-inhibitor. Such plants and genes aredescribed in WO 99/34008 and WO 02/36787. Tolerance of plants to HPPDinhibitors can also be improved by transforming plants with a geneencoding an enzyme having prephenate deshydrogenase (PDH) activity inaddition to a gene encoding an HPPD-tolerant enzyme, as described in WO2004/024928. Further, plants can be made more tolerant to HPPD-inhibitorherbicides by adding into their genome a gene encoding an enzyme capableof metabolizing or degrading HPPD inhibitors, such as the CYP450 enzymesshown in WO 2007/103567 and WO 2008/150473.

In particular embodiments, the invention discloses a cotton plant cell,plant part, plant, or seed comprising a chimeric gene comprising (a) anucleic acid sequence encoding a protein having HPPD activity, whereinsaid protein has a tryptophan at a position corresponding to position336 of SEQ ID NO: 19, wherein said protein provides to said planttolerance to a field dose of at least 1× of at least one HPPD inhibitor,operably linked to (b) a plant expressible promoter and optionally (c) atranslational termination and polyadenylation region.

It will be understood that the cotton plant cell, plant part, plant, orseed comprising a chimeric gene comprising (a) a nucleic acid sequenceencoding a protein having HPPD activity according to the invention doesnot need to be generated according to the methods described herein abovefor the targeted modification of the genome of a plant or plant cell,but may also be created by random transformation with the chimeric geneor by traditional breeding processes, as described elsewhere in thisapplication.

The term “a protein having HPPD activity” refers to a protein whichcatalyzes the reaction converting para-hydroxyphenylpyruvate(abbreviated herein as HPP), a tyrosine degradation product, intohomogentisate (abbreviated herein as HG).

The catalytic activity of a protein having HPPD activity may be definedby various methods well-known in the art. WO 2009/144079 describesvarious suitable screening methods. Initial screens may be performedwith chimeric genes comprising the nucleic acid encoding the HPPDprotein described herein being expressed in bacteria, such as acomplementation assay in e.g. E. coli (WO08/124495). Further and moreelaborate screens may be carried out in plant cells or plants expressingthe HPPD protein disclosed herein.

The same screenings may also be used when examining whether an HPPDprotein provides to a plant such as a cotton plant tolerance to a fielddose of at least 1× of at least one HPPD inhibitor as described furtherbelow, with the difference that said HPPD inhibitor is added in additionto an HPPD substrate. HPPD inhibitors which may be tested includeisoxaflutole, tembotrione, mesotrione, pyrasulfotole, bicyclopyrone,topramezone, tefuryltrione and sulcotrione and other HPPD inhibitorsmentioned in this application. A screening method which is simple toimplement is (a) to determine the dose of an HPPD inhibitor which doesnot inhibit the protein having HPPD activity according to the invention,such as that of SEQ ID NO: 3 which, e. g. for in vitro or cell cultureexperiments, should be a dose corresponding to a field dose of at least1×, at least 2× or even at least 3× or at least 4× of an HPPD inhibitorof choice; (b) to subject plant cells, plant parts or plants eachcomprising a chimeric gene according to the invention, wherein thenucleic acid encoding a protein having HPPD activity is a protein asdescribed above, to this dose, and thereafter (c) to isolate the plantcells, plant parts or plants which have withstood this otherwise lethaldose. At the same time or alternatively, the damage to the aerial partsof said plant parts or plants upon treatment with said inhibitor, suchas the extent of chlorosis, bleaching and/or necrosis, may be assessedand scored. Scoring may be effected e. g. as done on the appendedexamples or as described below.

A position corresponding to position 336 of SEQ ID NO: 19 refers to theamino acid sequence of HPPD proteins comprising an amino acid sequenceother than that of SEQ ID NO: 19, having a similar or the same overallstructure and, accordingly, also having an amino acid positioncorresponding to position 336 of SEQ ID NO: 19 but, depending on thelength of the amino acid sequence, possibly at a different position. Thecorresponding position in other HPPD proteins can be determined byaligning the sequences of these proteins with SEQ ID NO: 19 as describedabove.

In one example of the plant cell, plant part, plant or seed describedherein said nucleic acid sequence encoding a protein having HPPDactivity comprises the nucleotide sequence of SEQ ID NO: 20 from nt949-2025 or a nucleic acid sequence having at least 70%, at least 80%,at least 90%, at least 95%, at least 98% or at least 99% sequenceidentity thereto, wherein said protein provides to a plant tolerance toa field dose of at least 1× of at least one HPPD inhibitor. Sequencesfalling within this definition include those which encode the amino acidsequence of SEQ ID NO: 21 or an amino acid sequence with at least 70%,at least 80%, at least 90%, at least 95%, at least 98% or at least 99%sequence identity to SEQ ID NO:21.

Also included are fragments of said SEQ ID NO: 20 from nt 949-2025 aslong as the protein encoded thereby provides to a plant tolerance to afield dose of at least 1× of at least one HPPD inhibitor. Such fragmentscomprise for example at least 300, at least 400, at least 500, at least600, at least 700, at least 800, at least 900 or at least 1000nucleotides. In one example, any of the above described fragmentsinclude a nucleic acid sequence encoding an HPPD protein comprising atryptophan at a position equivalent to position 336 of SEQ ID NO: 19. Inanother example, said fragments include in addition at least the firstfive, first ten, first 20, first 30 amino acids N- and/or C-terminal ofsaid tryptophan.

Accordingly, in one example of the plant cell, plant part, plant or seeddescribed herein said amino acid sequence of a protein having HPPDactivity comprises the amino acid sequence of SEQ ID NO: 21 or an aminoacid sequence having at least 90%, at least 95%, at least 96%, at least97%, at least 98% or at least 99% sequence identity thereto, whereinsaid protein comprises the above amino acid substitution and provides tosaid plant tolerance to a field dose of at least 1× of at least one HPPDinhibitor. Also included are fragments of said SEQ ID NO: 21 as longsaid protein having HPPD activity comprises the above amino acidsubstitution and provides to a plant tolerance to a field dose of atleast 1× of at least one HPPD inhibitor. Such fragments comprise forexample at least 100 amino acids, at least 150 amino acids, at least 200amino acids, at least 250 amino acids, at least 300 amino acids.

As a regulatory sequence which functions as a promoter in plant cellsand plants, use may be made of any promoter sequence of a gene which isnaturally expressed in plants, in particular a promoter which isexpressed especially in the leaves of plants, such as for example“constitutive” promoters of bacterial, viral or plant origin.

A plant expressible promoter can be a constitutive promoter, i.e. apromoter capable of directing high levels of expression in most celltypes (in a spatio-temporal independent manner). Examples of plantexpressible constitutive promoters include promoters of bacterialorigin, such as the octopine synthase (OCS) and nopaline synthase (NOS)promoters from Agrobacterium, but also promoters of viral origin, suchas that of the cauliflower mosaic virus (CaMV) 35S transcript (Hapsteret al., 1988, Mol. Gen. Genet. 212: 182-190) or 19S RNAs genes (Odell etal., 1985, Nature. 6; 313(6005):810-2; U.S. Pat. No. 5,352,605; WO84/02913; Benfey et al., 1989, EMBO J. 8:2195-2202), the enhanced 2×35Spromoter (Kay at al., 1987, Science 236:1299-1302; Dada et al. (1993),Plant Sci 94:139-149) promoters of the cassava vein mosaic virus (CsVMV;WO 97/48819, U.S. Pat. No. 7,053,205), 2×CsVMV (WO2004/053135) thecircovirus (AU 689 311) promoter, the sugarcane bacilliform badnavirus(ScBV) promoter (Samac et al., 2004, Transgenic Res. 13(4):349-61), thefigwort mosaic virus (FMV) promoter (Sanger et al., 1990, Plant MolBiol. 14(3):433-43), the subterranean clover virus promoter No 4 or No 7(WO 96/06932) and the enhanced 35S promoter as described in U.S. Pat.No. 5,164,316, U.S. Pat. No. 5,196,525, U.S. Pat. No. 5,322,938, U.S.Pat. No. 5,359,142 and U.S. Pat. No. 5,424,200. Among the promoters ofplant origin, mention will be made of the promoters of the plantribulose-biscarboxylase/oxygenase (Rubisco) small subunit promoter (U.S.Pat. No. 4,962,028; WO99/25842) from zea mays and sunflower, thepromoter of the Arabidopsis thaliana histone H4 gene (Chabouté et at,1987), the ubiquitin promoters (Holtorf et al., 1995, Plant Mol. Biol.29:637-649, U.S. Pat. No. 5,510,474) of Maize, Rice and sugarcane, theRice actin 1 promoter (Act-1, U.S. Pat. No. 5,641,876), the histonepromoters as described in EP 0 507 698 A1, the Maize alcoholdehydrogenase 1 promoter (Adh-1) (fromhttp://www.patentlens.net/daisy/promoters/242.html)). Also the smallsubunit promoter from Chrysanthemum may be used if that use is combinedwith the use of the respective terminator (Outchkourov et al., Planta,216: 1003-1012, 2003).

Alternatively, a promoter sequence specific for particular regions,tissues or organs of plants can be used to express the HPPD proteindisclosed herein. Promoters that can be used are tissue-specific ororgan-specific promoters like organ primordia-specific promoters (An etal., 1996, Plant Cell 8: 15-30), stem-specific promoters (Keller et al.,1988, EMBO J. 7(12): 3625-3633), mesophyl-specific promoters (such asthe light-inducible Rubisco promoters), root-specific promoters (Kelleret al., 1989, Genes Dev. 3: 1639-1646), vascular tissue-specificpromoters (Peleman et al., 1989, Gene 84: 359-369), and the like.

Use may also be made of an inducible promoter advantageously chosen fromthe phenylalanine ammonia lyase (PAL), HMG-CoA reductase (HMG),chitinase, glucanase, proteinase inhibitor (PI), PRI family gene,nopaline synthase (nos) and vspB promoters (U.S. Pat. No. 5,670,349,Table 3), the HMG2 promoter (U.S. Pat. No. 5,670,349), the applebeta-galactosidase (ABGI) promoter and the apple aminocyclopropanecarboxylate synthase (ACC synthase) promoter (WO 98/45445).

According to the invention, use may also be made, in combination withthe promoter, of other regulatory sequences, which are located betweenthe promoter and the coding sequence, such as transcription activators(“enhancers”), for instance the translation activator of the tobaccomosaic virus (TMV) described in Application WO 87/07644, or of thetobacco etch virus (TEV) described by Carrington & Freed 1990, J. Virol.64: 1590-1597, for example.

Other regulatory sequences that enhance functional expression andthereby herbicide tolerance may also be located within the chimericgene. One example of such regulatory sequences are introns. Introns areintervening sequences present in the pre-mRNA but absent in the matureRNA following excision by a precise splicing mechanism. The ability ofnatural introns to enhance gene expression, a process referred to asintron-mediated enhancement (IME), has been known in various organisms,including mammals, insects, nematodes and plants (WO 07/098042, p11-12). IME is generally described as a posttranscriptional mechanismleading to increased gene expression by stabilization of the transcript.The intron is required to be positioned between the promoter and thecoding sequence in the normal orientation. However, some introns havealso been described to affect translation, to function as promoters oras position and orientation independent transcriptional enhancers(Chaubet-Gigot et al., 2001, Plant Mol Biol. 45(1):17-30, p 27-28).

Examples of genes containing such introns include the 5′ introns fromthe rice actin 1 gene (see U.S. Pat. No. 5,641,876), the rice actin 2gene, the maize sucrose synthase gene (Clancy and Hannah, 2002, PlantPhysiol. 130(2):918-29), the maize alcohol dehydrogenase-1 (Adh-1) andBronze-1 genes (Callis et al. 1987 Genes Dev. 1(10):1183-200;Mascarenhas et al. 1990, Plant Mol Biol. 15(6):913-20), the maize heatshock protein 70 gene (see U.S. Pat. No. 5,593,874), the maize shrunken1 gene, the light sensitive 1 gene of Solanum tuberosum, and the heatshock protein 70 gene of Petunia hybrida (see U.S. Pat. No. 5,659,122),the replacement histone H3 gene from alfalfa (Keleman et al. 2002Transgenic Res. 11(1):69-72) and either replacement histone H3 (histoneH3.3-like) gene of Arabidopsis thaliana (Chaubet-Gigot et al., 2001,Plant Mol Biol. 45(1):17-30).

Other suitable regulatory sequences include 5′ UTRs. As used herein, a5′UTR, also referred to as leader sequence, is a particular region of amessenger RNA (mRNA) located between the transcription start site andthe start codon of the coding region. It is involved in mRNA stabilityand translation efficiency. For example, the 5′ untranslated leader of apetunia chlorophyll a/b binding protein gene downstream of the 35Stranscription start site can be utilized to augment steady-state levelsof reporter gene expression (Harpster et al., 1988, Mol Gen Genet.212(1):182-90). WO95/006742 describes the use of 5′ non-translatedleader sequences derived from genes coding for heat shock proteins toincrease transgene expression.

The chimeric gene may also comprise a transcription termination orpolyadenylation sequence operable in plant cells. As a transcriptiontermination or polyadenylation sequence, use may be made of anycorresponding sequence of bacterial origin, such as for example the nosterminator of Agrobacterium tumefaciens, of viral origin, such as forexample the CaMV 35S terminator, or of plant origin, such as for examplea histone terminator as described in published Patent Application EP 0633 317 A1.

In connection with the present application, the expression of chimericgenes conferring tolerance to at least one HPPD inhibitor in cotton maybe further enhanced by optimizing the sequence encoding the protein tobe expressed in cotton, thereby taking into account, inter alia, thecodon usage of cotton. Accordingly, the nucleic acid sequence encodingthe HPPD protein disclosed herein may be optimized for expression incotton, e. g. by codon optimization (available e. g. viawww.entelechon.com).

An example of a cotton codon-optimized nucleic acid sequence encoding aprotein having HPPD activity disclosed herein is represented by SED IDNo. 20 from nt 949-2025. The protein having HPPD activity disclosedherein may be encoded by the nucleic acid sequence of SEQ ID NO: 20 fromnt 949-2025 or have the amino acid sequence of SEQ ID NO: 21. In oneexample, said chimeric gene comprises or consists of the nucleic acidsequence of SEQ ID NO: 20 from position 88 to position 2714.

Tolerance to at least one HPPD inhibitor as observed in the plant cell,plant part, plant or seed disclosed herein is caused by the proteinhaving HPPD activity disclosed herein and introduced into said plantcell, plant part, plant which provides to said plant cell, plant part,plant or seed said tolerance to at least one HPPD inhibitor.

The terms “tolerance” or “tolerant” denote the reduced or complete lackof susceptibility of a plant expressing the protein having HPPD activitydisclosed herein to substances, particularly herbicides, which inhibitHPPD proteins, optionally in comparison with the plant's own HPPDprotein. More specifically, said terms mean the relative levels ofinherent tolerance, i.e. reduced or complete lack of susceptibility asdescribed above, of the protein having HPPD activity screened accordingto a visible indicator phenotype of the strain or plant transformed witha nucleic acid comprising the gene coding for the respective protein inthe presence of different concentrations of an HPPD inhibitor. The HPPDinhibitor may be selected from any available HPPD inhibitors.

Usually, application of an HPPD inhibitor to plants not expressing anHPPD enzyme providing tolerance to said HPPD inhibitor results inserious damage of the aerial parts of the plant shortly afterapplication, whereas no or only minor damage is observed in plantsexpressing an HPPD protein according to the invention or other HPPDproteins believed to provide tolerance. Accordingly, the toleranceobserved for said at least one HPPD inhibitor provides plants expressingthe HPPD protein according to the invention and treated with at leastone HPPD inhibitor with agronomical advantages as compared to plants notexpressing said HPPD protein or expressing a different HPPD protein.Although the plants of the invention may show some minor damage afterherbicide treatment, said plants recover a short time after treatment.Therefore, plants expressing the HPPD protein of the invention, whentreated, preferably do not have a reduced agronomical performance, e.g.do not display a reduced yield, compared to untreated plants.

At present, most commercially available HPPD inhibitors are attributedto one of these four chemical families:

1) the triketones, e.g. sulcotrione [i.e.2-[2-chloro-4-(methylsulfonyl)benzoyl]-1,3-cyclohexanedione], mesotrione[i.e.2-[4-(methylsulfonyl)-2-nitrobenzoyl]-1,3-cyclohexanedione];tembotrione[i.e.2-[2-chloro-4-(methylsulfonyl)-3-[(2,2,2,-tri-fluoroethoxy)methyl]benzoyl]-1,3-cyclohexanedione];tefuryltrione [i.e.2-[2-chloro-4-(methylsulfonyl)-3-[[(tetrahydro-2-furanyl)methoxy]methyl]benzoyl]-1,3cyclohexanedione]]; bicyclopyrone [i.e.4-hydroxy-3-[[2-[(2-methoxyethoxy)methyl]-6-(trifluoromethyl)-3-pyridinyl]carbonyl]bicyclo[3.2.1]oct-3-en-2-one];benzobicyclone [i.e.3-(2-chloro-4-mesylbenzoyl)-2-phenylthiobicyclo[3.2.1]oct-2-en-4-one];

2) The isoxazoles, e.g. isoxaflutole [i.e.(5-cyclopropyl-4-isoxazolyl)[2-(methylsulfonyl)-4-(trifluoromethyl)phenyl]methanone]or corresponding diketonitriles; and

3) the pyrazolinones, e.g. topramezone[i.e.[3-(4,5-dihydro-3-isoxazolyl)-2-methyl-4-(methylsulfonyl)phenyl](5-hydroxy-1-methyl-1H-pyrazol-4-yl)methanone], pyrasulfotole[(5-hydroxy-1,3-dimethylpyrazol-4-yl(2-mesyl-4trifluaromethylphenyl)methanone];pyrazoxyfen[2-[4-(2,4-dichlorobenzoyl)-1,3-dimethylpyrazol-5-yloxy]acetophenone].

Further compound classes of HPPD inhibitors useful in the presentinvention are the N-(tetrazol-5-yl)- andN-(triazol-5-yl)arylcarboxamides as disclosed in PCT/EP2011/064820 andthe N-(1,2,5-Oxadiazol-3-yl)benzamides as disclosed in WO2011/035874.

In one example, the at least one HPPD inhibitor is selected fromisoxaflutole, tembotrione, mesotrione, pyrasulfotole and topramezone orany other applicable HPPD inhibitor or HPPD herbicide such as the oneslisted herein.

Dose responses and relative shifts in dose responses associated withthese indicator phenotypes (growth inhibition, chlorosis, bleaching,leaf damage in general, wilting, necrosis, herbicidal effect etc) areconveniently expressed in terms, for example, of GR50 (concentration for50% reduction of growth) or MIC (minimum inhibitory concentration)values where increases in values correspond to increases in inherenttolerance of the expressed HPPD, in the normal manner based upon plantdamage (such as damage of the aerial parts of the plant, which maymanifest in bleaching, chlorosis and/or necrosis), meristematicbleaching symptoms etc. at a range of different concentrations ofherbicides. These data can be expressed in terms of, for example, GR50values derived from dose/response curves having “dose” plotted on thex-axis and “percentage kill”, “herbicidal effect”, “numbers of emerginggreen plants” etc. plotted on the y-axis where increased GR50 valuescorrespond to increased levels of inherent tolerance of the expressedHPPD. Herbicides can be applied pre-emergence or post-emergence, asdescribed in the appended examples. Alternatively, plants can beevaluated for damage of their green parts or leaves based on therelevant indicator phenotypes as mentioned above, e.g. on a scale of0-100, where 0% indicates no damage and 100% indicated completebleaching, chlorosis, necrosis and/or wilting etc.

As used herein “pre-emergence”, refers to the application of aherbicide, e.g. the at least one HPPD inhibitor, prior to the emergenceabove the surface of the soil or growth medium of the seedlings grownfrom the sowed seeds, e.g. just prior to or at the time of sowing of theseeds, or just after sowing, to the soil or growth medium wherein theseeds are sown. As used herein “post-emergence”, refers to theapplication of the herbicide to the plant and the soil or growth mediumafter the emergence of the seedlings, for instance at the 2-3 leaf-stageor later.

The plant cell, plant part, plant or seed comprising the chimeric HPPDgene as disclosed herein is tolerant to a field dose of at least 1× ofat least one HPPD inhibitor. The at least one HPPD inhibitor can beapplied at the pre-emergence stage and/or at the post-emergence stage.

As used herein “1×”, refers to a normal, single field dose of anHPPD-inhibitor, such as an HPPD-inhibiting herbicide or a formulationcomprising an HPPD inhibitor, indicated in g a.i./ha, whereby a.i.stands for active ingredient, as commercially used. Field doses maydiffer depending on e. g. the HPPD inhibitor used, the crop to which itis applied and the conditions of application, e. g the type of soil onwhich the crop is grown and the weed expected to be present. Field dosesof 1× of commercial products are stated on the product label. Dependingon product and label recommendations, commercial 1× field rates may varybetween e.g. 18.41 g a.i./ha for topramezone as present in the productImpact™ and e.g. 138 g a.i./ha for tembotrione as present in the productLaudis SC™.

In the field trials in the present examples, mesotrione present in theproduct Callisto™ was applied in a field dose of 1× corresponding to 105g a.i./ha, and topramezone as present in the product Impact™ was appliedin a field dose of 1× corresponding to 18.41 g a.i./ha.

Further exemplary 1× field doses for selected HPPD inhibitors areIsoxaflutole (Balance Flexx™): 105 g a.i./ha

Pyrasulfutole: 37.5 g a.i./ha

Tembotrione(2-{2-chloro-4-mesyl-3-[(2,2,2-trifluoroethoxy)methyl]benzoyl}cyclohexane-1,3-dioneor2-[2-chloro-4-(methylsulfonyl)-3-[(2,2,2-trifluoroethoxy)methyl]benzoyl]-1,3-cyclohexanedione;formulation Laudis SC™): 138 g a.i./ha.

Another exemplary 1× field dose of tembotrione: 100 g a.i./ha.Accordingly, a field dose of 1.5× refers to an applied dose of 150% ofthe 1× dose, a field dose of 2× refers to an applied dose of 200% of the1× dose of a specific HPPD inhibitor or HPPD inhibitor formulation andso on.

In another example, the plant cell, plant part, plant or seed disclosedherein has tolerance to a field dose of at least 2× of at least one HPPDinhibitor.

In yet a further example, the plant cell, plant part, plant or seeddisclosed herein has tolerance to a field dose of at least 3× or atleast 4× of at least one HPPD inhibitor.

In a particular embodiment, the plant cell, plant part, plant or seeddisclosed herein has tolerance to a field dose of at least 1×IFT, of atleast 2×IFT or of at least 4×IFT. In an even further embodiment, saiddose of at least 1×IFT, of at least 2×IFT, or of at least 4×IFT isapplied pre-emergence.

Tolerance to a particular dose of an HPPD inhibitor such as an HPPDinhibitor herbicide, as used herein, refers to a plant displaying aminimal amount of damage after treatment with said herbicide dose.Accordingly, a herbicide tolerant plant according to the invention, inthe field, shows a visual damage of the aerial parts of the plant of notmore than 40%, not more than 35%, not more than 30%, not more than 27%,not more than 25%, not more than 20%, not more than 15%, not more than10%, not more than 9%, not more than 8%, not more than 7%, not more than6% or not more than 5% seven days after treatment with at least one HPPDinhibitor. Damage may be assessed by the extent of bleaching, chlorosisand/or necrosis of the aerial parts of the plant, including leaves andstem. Accordingly, tolerance may manifest in chlorosis, bleaching and/ornecrosis of not more than 40%, not more than 35%, not more than 30%, notmore than 27%, not more than 25%, not more than 20%, not more than 15%,not more than 10%, not more than 9%, not more than 8%, not more than 7%,not more than 6% or not more than 5% seven days after treatment with atleast one HPPD inhibitor. Damage or plant response scoring is describedelsewhere in the application.

Alternatively, a herbicide tolerant plant according to the invention, inthe field, shows a visual damage of the aerial parts of the plant of notmore than 20%, not more than 15%, not more than 10%, not more than 9%,not more than 8%, not more than 7%, not more than 6% or not more than 5%21 days after treatment with at least one HPPD inhibitor. In other wordsand in accordance with the above, tolerance may manifest in chlorosis,bleaching and/or necrosis of not more than 20%, not more than 15%, notmore than 10° A, not more than 9%, not more than 8%, not more than 7%,not more than 6% or not more than 5% 21 days after treatment with atleast one HPPD inhibitor.

In a different alternative, a herbicide tolerant plant according to theinvention, in the field, shows a visual damage of the aerial parts ofthe plant of not more than 10° A, not more than 6%, not more than 5%,not more than 4% or not more than 3% 21 days after treatment with atleast one HPPD inhibitor. In other words and in accordance with theabove, tolerance may manifest in chlorosis, bleaching and/or necrosis ofnot more than 10%, not more than 6%, not more than 5%, not more than 4%or not more than 3% 21 days after treatment with at least one HPPDinhibitor.

Said at least one HPPD inhibitor can also be more than one HPPDinhibitor such as at least two, at least three, at least, four, at leastfive, at least six, at least 10 or even more HPPD inhibitors. Thereby,any combination of HPPD inhibitors for each of the above options ispossible. For example, plants of the invention may be tolerant to atleast two HPPD inhibitors or the chimeric gene of the invention mayconfer tolerance to at least two HPPD inhibitors, wherein said at leasttwo inhibitors may comprise isoxaflutole and topramezone, isoxaflutoleand mesotrione, isoxaflutole and pyrasulfutole, isoxaflutole andtembotrione, topramezone and mesotrione, topramezone and pyrasulfutole,topramezone and tembotrione, mesotrione and pyrasulfutole, mesotrioneand tembotrione or pyrasulfultole and tembotrione. Or said at least oneHPPD inhibitor may be at least three HPPD inhibitors, wherein said atleast three inhibitors may comprise isoxaflutole, topramezone andmesotrione; isoxaflutole, topramezone and pyrasulfutole; isoxaflutole,topramezone and tembotrione; isoxaflutole, mesotrione and pyrasulfutole;isoxaflutole, mesotrione and tembotrione; isoxaflutole, mesotrione andpyrasulfutole; isoxaflutole, isoxaflutole and tembotrione; topramezone,mesotrione and pyrasulfutol; topramezone, mesotrione and tembotrione;topramezone, pyrasulfutole and tembotrione or mesotrione, pyrasulfutoleand tembotrione. Combinations of at least four HPPD inhibitors maycomprise isoxaflutole, topramezone, mesotrione and pyrasulfutole;isoxaflutole, topramezone, mesotrione and tembotrione; isoxaflutole,mesotrione, pyrasulfutole and tembotrione or topramezone, mesotrione,pyrasulfutole and tembotrione. An exemplary combination of at least fiveHPPD inhibitors comprises comprise isoxaflutole, topramezone,mesotrione, pyrasulfutole and tembotrione.

Tolerance levels to different HPPD inhibitors may also differ. Forexample, whereas the chimeric gene of the invention confers tolerance tocotton plants to 2× for one HPPD inhibitor, it may be 1× for anotherone. In any case, tolerance is provided to at least 1× of each HPPDinhibitor as claimed.

Other herbicide-tolerance genes include a gene encoding the enzyme5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Examples of suchEPSPS genes are the AroA gene (mutant CT7) of the bacterium Salmonellatyphimurium (Comai et al., 1983, Science 221, 370-371), the CP4 gene ofthe bacterium Agrobacterium sp. (Barry et al., 1992, Curr. Topics PlantPhysiol. 7, 139-145), the genes encoding a Petunia EPSPS (Shah et al.,1986, Science 233, 478-481), a Tomato EPSPS (Gasser et al., 1988, J.Biol. Chem. 263, 4280-4289), or an Eleusine EPSPS (WO 01/66704). It canalso be a mutated EPSPS as described in for example EP 0837944, WO00/66746, WO 00/66747 or WO02/26995. Glyphosate-tolerant plants can alsobe obtained by expressing a gene that encodes a glyphosateoxido-reductase enzyme as described in U.S. Pat. Nos. 5,776,760 and5,463,175. Glyphosate-tolerant plants can also be obtained by expressinga gene that encodes a glyphosate acetyl transferase enzyme as describedin for example WO 02/36782, WO 03/092360, WO 05/012515 and WO 07/024782.Glyphosate-tolerant plants can also be obtained by selecting plantscontaining naturally-occurring mutations of the above-mentioned genes,as described in for example WO 01/024615 or WO 03/013226. EPSPS genesthat confer glyphosate tolerance are described in e.g. U.S. patentapplication Ser. Nos. 11/517,991, 10/739,610, 12/139,408, 12/352,532,11/312,866, 11/315,678, 12/421,292, 11/400,598, 11/651,752, 11/681,285,11/605,824, 12/468,205, 11/760,570, 11/762,526, 11/769,327, 11/769,255,11/943801 or Ser. No. 12/362,774. Other genes that confer glyphosatetolerance, such as decarboxylase genes, are described in e.g. U.S.patent application Ser. Nos. 11/588,811, 11/185,342, 12/364,724,11/185,560 or Ser. No. 12/423,926.

Among the DNA sequences encoding a suitable EPSPS which confer toleranceto the herbicides which have EPSPS as a target, mention will moreparticularly be made of the gene which encodes a plant EPSPS, inparticular maize EPSPS, particularly a maize EPSPS which comprises twomutations, particularly a mutation at amino acid position 102 and amutation at amino acid position 106 (WO 2004/074443), and which isdescribed in U.S. Pat. No. 6,566,587, hereinafter named double mutantmaize EPSPS or 2mEPSPS (see also SEQ ID NO: 20 from nt 4602-5939 for anucleic acid sequence encoding this EPSPS and SEQ ID NO: 22 for theamino acid sequence of the resulting enzyme).

In the case of the DNA sequences encoding EPSPS, and more particularlyencoding the above genes, the sequence encoding these enzymes isadvantageously preceded by a sequence encoding a transit peptidetargeting the EPSPS protein to the chloroplast. A particular chloroplasttransit peptide of interest to express EPSPS proteins is the “optimizedtransit peptide” as described in U.S. Pat. No. 5,510,471 or 5,633,448.

Even other herbicide tolereance genes may encode an enzyme detoxifyingthe herbicide or a mutant glutamine synthase enzyme that is resistant toinhibition, e.g. described in U.S. patent application Ser. No.11/760,602. One such efficient detoxifying enzyme is an enzyme encodinga phosphinothricin acetyltransferase (such as the bar or pat proteinfrom Streptomyces species). Phosphinothricin acetyltransferases are forexample described in U.S. Pat. Nos. 5,561,236; 5,648,477; 5,646,024;5,273,894; 5,637,489; 5,276,268; 5,739,082; 5,908,810 and 7,112,665.

Still further herbicide tolerance genes encode variant ALS enzymes (alsoknown as acetohydroxyacid synthase, AHAS) as described for example inTranel and Wright (2002, Weed Science 50:700-712), but also, in U.S.Pat. Nos. 5,605,011, 5,378,824, 5,141,870, and 5,013,659. The productionof sulfonylurea-tolerant plants and imidazolinone-tolerant plants isdescribed in U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141,870; 5,767,361;5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937; and 5,378,824;and international publication WO 96/33270. Other imidazolinone-tolerancegenes are also described in for example WO 2004/040012, WO 2004/106529,WO 2005/020673, WO 2005/093093, WO 2006/007373, WO 2006/015376, WO2006/024351, and WO 2006/060634. Further sulfonylurea- andimidazolinone-tolerance genes are described in for example WO 07/024782and U.S. Patent Application No. 61/288,958.

Insect resistance gene may comprise a coding sequence encoding:

1) an insecticidal crystal protein from Bacillus thuringiensis or aninsecticidal portion thereof, such as the insecticidal crystal proteinslisted by Crickmore et al. (1998, Microbiology and Molecular BiologyReviews, 62: 807-813), updated by Crickmore et al. (2005) at theBacillus thuringiensis toxin nomenclature, online at:http://www.lifesci.sussex.ac.uk/Home/Neil_Crickmore/Bt/), orinsecticidal portions thereof, e.g., proteins of the Cry protein classesCry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1F, Cry2Ab, Cry3Aa, or Cry3Bb orinsecticidal portions thereof (e.g. EP 1999141 and WO 2007/107302), orsuch proteins encoded by synthetic genes as e.g. described in and U.S.patent application Ser. No. 12/249,016; or2) a crystal protein from Bacillus thuringiensis or a portion thereofwhich is insecticidal in the presence of a second other crystal proteinfrom Bacillus thuringiensis or a portion thereof, such as the binarytoxin made up of the Cry34 and Cry35 crystal proteins (Moellenbeck etal. 2001, Nat. Biotechnol. 19: 668-72; Schnepf et al. 2006, AppliedEnvironm. Microbiol. 71, 1765-1774) or the binary toxin made up of theCry1A or Cry1F proteins and the Cry2Aa or Cry2Ab or Cry2Ae proteins(U.S. patent application Ser. No. 12/214,022 and EP 08010791.5); or3) a hybrid insecticidal protein comprising parts of differentinsecticidal crystal proteins from Bacillus thuringiensis, such as ahybrid of the proteins of 1) above or a hybrid of the proteins of 2)above, e.g., the Cry1A.105 protein produced by corn event MON89034 (WO2007/027777); or4) a protein of any one of 1) to 3) above wherein some, particularly 1to 10, amino acids have been replaced by another amino acid to obtain ahigher insecticidal activity to a target insect species, and/or toexpand the range of target insect species affected, and/or because ofchanges introduced into the encoding DNA during cloning ortransformation, such as the Cry3Bb1 protein in corn events MON863 orMON88017, or the Cry3A protein in corn event MIR604; or5) an insecticidal secreted protein from Bacillus thuringiensis orBacillus cereus, or an insecticidal portion thereof, such as thevegetative insecticidal (VIP) proteins listed at:http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html, e.g.,proteins from the VIP3Aa protein class; or6) a secreted protein from Bacillus thuringiensis or Bacillus cereuswhich is insecticidal in the presence of a second secreted protein fromBacillus thuringiensis or B. cereus, such as the binary toxin made up ofthe VIP1A and VIP2A proteins (WO 94/21795); or7) a hybrid insecticidal protein comprising parts from differentsecreted proteins from Bacillus thuringiensis or Bacillus cereus, suchas a hybrid of the proteins in 1) above or a hybrid of the proteins in2) above; or8) a protein of any one of 5) to 7) above wherein some, particularly 1to 10, amino acids have been replaced by another amino acid to obtain ahigher insecticidal activity to a target insect species, and/or toexpand the range of target insect species affected, and/or because ofchanges introduced into the encoding DNA during cloning ortransformation (while still encoding an insecticidal protein), such asthe VIP3Aa protein in cotton event COT102; or9) a secreted protein from Bacillus thuringiensis or Bacillus cereuswhich is insecticidal in the presence of a crystal protein from Bacillusthuringiensis, such as the binary toxin made up of VIP3 and Cry1A orCry1F (U.S. Patent Appl. No. 61/126,083 and 61/195019), or the binarytoxin made up of the VIP3 protein and the Cry2Aa or Cry2Ab or Cry2Aeproteins (U.S. patent application Ser. No. 12/214,022 and EP08010791.5);10) a protein of 9) above wherein some, particularly 1 to 10, aminoacids have been replaced by another amino acid to obtain a higherinsecticidal activity to a target insect species, and/or to expand therange of target insect species affected, and/or because of changesintroduced into the encoding DNA during cloning or transformation (whilestill encoding an insecticidal protein).An “insect-resistant gene as used herein, further includes transgenescomprising a sequence producing upon expression a double-stranded RNAwhich upon ingestion by a plant insect pest inhibits the growth of thisinsect pest, as described e.g. in WO 2007/080126, WO 2006/129204, WO2007/074405, WO 2007/080127 and WO 2007/035650.

Abiotic Stress Tolerance Genes Include

1) a transgene capable of reducing the expression and/or the activity ofpoly(ADP-ribose) polymerase (PARP) gene in the plant cells or plants asdescribed in WO 00/04173, WO/2006/045633, EP 04077984.5, or EP06009836.5.2) a transgene capable of reducing the expression and/or the activity ofthe PARG encoding genes of the plants or plants cells, as described e.g.in WO 2004/090140.3) a transgene coding for a plant-functional enzyme of the nicotineamideadenine dinucleotide salvage synthesis pathway including nicotinamidase,nicotinate phosphoribosyltransferase, nicotinic acid mononucleotideadenyl transferase, nicotinamide adenine dinucleotide synthetase ornicotine amide phosphorybosyltransferase as described e.g. in EP04077624.7, WO 2006/133827, PCT/EP07/002433, EP 1999263, or WO2007/107326.

Enzymes involved in carbohydrate biosynthesis include those described ine.g. EP 0571427, WO 95/04826, EP 0719338, WO 96/15248, WO 96/19581, WO96/27674, WO 97/11188, WO 97/26362, WO 97/32985, WO 97/42328, WO97/44472, WO 97/45545, WO 98/27212, WO 98/40503, WO99/58688, WO99/58690, WO 99/58654, WO 00/08184, WO 00/08185, WO 00/08175, WO00/28052, WO 00/77229, WO 01/12782, WO 01/12826, WO 02/101059, WO03/071860, WO 2004/056999, WO 2005/030942, WO 2005/030941, WO2005/095632, WO 2005/095617, WO 2005/095619, WO 2005/095618, WO2005/123927, WO 2006/018319, WO 2006/103107, WO 2006/108702, WO2007/009823, WO 00/22140, WO 2006/063862, WO 2006/072603, WO 02/034923,EP 06090134.5, EP 06090228.5, EP 06090227.7, EP 07090007.1, EP07090009.7, WO 01/14569, WO 02/79410, WO 03/33540, WO 2004/078983, WO01/19975, WO 95/26407, WO 96/34968, WO 98/20145, WO 99/12950, WO99/66050, WO 99/53072, U.S. Pat. No. 6,734,341, WO 00/11192, WO98/22604, WO 98/32326, WO 01/98509, WO 01/98509, WO 2005/002359, U.S.Pat. No. 5,824,790, U.S. Pat. No. 6,013,861, WO 94/04693, WO 94/09144,WO 94/11520, WO 95/35026 or WO 97/20936 or enzymes involved in theproduction of polyfructose, especially of the inulin and levan-type, asdisclosed in EP 0663956, WO 96/01904, WO 96/21023, WO 98/39460, and WO99/24593, the production of alpha-1,4-glucans as disclosed in WO95/31553, US 2002031826, U.S. Pat. No. 6,284,479, U.S. Pat. No.5,712,107, WO 97/47806, WO 97/47807, WO 97/47808 and WO 00/14249, theproduction of alpha-1,6 branched alpha-1,4-glucans, as disclosed in WO00/73422, the production of alternan, as disclosed in e.g. WO 00/47727,WO 00/73422, EP 06077301.7, U.S. Pat. No. 5,908,975 and EP 0728213, theproduction of hyaluronan, as for example disclosed in WO 2006/032538, WO2007/039314, WO 2007/039315, WO 2007/039316, JP 2006304779, and WO2005/012529.

Cotton plants or plant cultivars (that can be obtained by plantbiotechnology methods such as genetic engineering) which may also betreated according to the invention are plants, such as cotton plants,with altered fiber characteristics. Such plants can be obtained bygenetic transformation, or by selection of plants contain a mutationimparting such altered fiber characteristics and include:

-   a) Plants, such as cotton plants, containing an altered form of    cellulose synthase genes as described in WO 98/00549-   b) Plants, such as cotton plants, containing an altered form of rsw2    or rsw3 homologous nucleic acids as described in WO 2004/053219-   c) Plants, such as cotton plants, with increased expression of    sucrose phosphate synthase as described in WO 01/17333-   d) Plants, such as cotton plants, with increased expression of    sucrose synthase as described in WO 02/45485-   e) Plants, such as cotton plants, wherein the timing of the    plasmodesmatal gating at the basis of the fiber cell is altered,    e.g. through downregulation of fiber-selective β-1,3-glucanase as    described in WO 2005/017157, or as described in EP 08075514.3 or    U.S. Patent Appl. No. 61/128,938-   f) Plants, such as cotton plants, having fibers with altered    reactivity, e.g. through the expression of    N-acetylglucosaminetransferase gene including nodC and chitin    synthase genes as described in WO 2006/136351.

It is also an embodiment of the invention to provide chimeric genesencoding re-designed meganucleases as herein described, wherein thechimeric gene comprise a plant expressible promoter operably linked to aDNA region encoding a protein comprising an amino acid sequencecorresponding to the amino acid sequence of I-CreI as a scaffold (SEQ IDNO 16) comprising an S at position 32; Y at position 33; Q at position38; Q at position 80; S at position 40; T at position 42; R at position77; Y at position 68; Q at position 70; H at position 75; T at position44; I at position 24; Q at position 26; K at position 28 and an N atposition 30, and/or wherein the chimeric gene comprise a plantexpressible promoter operably linked to a DNA region encoding a secondprotein protein comprising an amino acid sequence corresponding to theamino acid sequence of I-CreI as a scaffold (SEQ ID NO 16) comprising anS at position 70; Q at position 44; K at position 24; A at position 26;K at position 28; N at position 30; S at position 32; Y at position 33;Q at position 38; Q at position 80; S at position 40; T at position 42;Q at position 77 and a Y at position 68, such as a protein comprisingthe amino acid sequence of SEQ ID 5 or SEQ ID 6 (corresponding aminoacid positions in redesigned meganucleases with respect to I-CreI can bedetermined by alignment).

The person skilled in the art will appreciate that, in addition to thenuclear genome, the methods of the invention may also be applied tomodify e.g. the chloroplast genome or mitochondrial genome, whereby DSBinduction at the predefined site and can further be enhanced byproviding the correct targeting signal to the endonuclease enzyme.

It will be appreciated that the methods of the invention can be appliedto any plant (Angiospermae or Gymnospermae) including but not limited tocotton, canola, oilseed rape, soybean, vegetables, potatoes, Lemna spp.,Nicotiana spp., Arabidopsis, alfalfa, barley, bean, corn, cotton, flax,millet, pea, rape, rice, rye, safflower, sorghum, soybean, sunflower,tobacco, turfgrass, wheat, asparagus, beet and sugar beet, broccoli,cabbage, carrot, cauliflower, celery, cucumber, eggplant, lettuce,onion, oilseed rape, pepper, potato, pumpkin, radish, spinach, squash,sugar cane, tomato, zucchini, almond, apple, apricot, banana,blackberry, blueberry, cacao, cherry, coconut, cranberry, date, grape,grapefruit, guava, kiwi, lemon, lime, mango, melon, nectarine, orange,papaya, passion fruit, peach, peanut, pear, pineapple, pistachio, plum,raspberry, strawberry, tangerine, walnut and watermelon.

In a specific embodiment, the plant is cotton. Cotton, as used hereinrefers to any existing cotton variety. For example, the cotton plantcell can be from a variety useful for growing cotton. The most commonlyused cotton varieties are Gossypium barbadense, G. hirsutum, G. arboreumand G. herbaceum. Further varieties include G. africanum and G.raimondii. The same applies to the cotton plant, cotton plant part andthe cotton seed described herein.

Example cotton plants disclosed herein include those from whichembryogenic callus can be derived, such as Coker 312, Coker310, Coker5Acala SJ-5, GSC25110, FIBERMAX 819, Siokra 1-3, T25, GSA75, Acala SJ2,Acala SJ4, Acala SJ5, Acala SJ-C1, Acala B1644, Acala B1654-26, AcalaB1654-43, Acala B3991, Acala GC356, Acala GC510, Acala GAM1, Acala C1,Acala Royale, Acala Maxxa, Acala Prema, Acala B638, Acala B1810, AcalaB2724, Acala B4894, Acala B5002, non Acala “picker” Siokra, “stripper”variety FC2017, Coker 315, STONEVILLE 506, STONEVILLE 825, DP50, DP61,DP90, DP77, DES119, McN235, HBX87, HBX191, HBX107, FC 3027, CHEMBRED A1,CHEMBRED A2, CHEMBRED A3, CHEMBRED A4, CHEMBRED B1, CHEMBRED B2,CHEMBRED B3, CHEMBRED C1, CHEMBRED C2, CHEMBRED C3, CHEMBRED C4,PAYMASTER 145, HS26, HS46, SICALA, PIMA S6 ORO BLANCO PIMA, FIBERMAXFM5013, FIBERMAX FM5015, FIBERMAX FM5017, FIBERMAX FM989, FIBERMAXFM832, FIBERMAX FM966, FIBERMAX FM958, FIBERMAX FM989, FIBERMAX FM958,FIBERMAX FM832, FIBERMAX FM991, FIBERMAX FM819, FIBERMAX FM800, FIBERMAXFM960, FIBERMAX FM966, FIBERMAX FM981, FIBERMAX FM5035, FIBERMAX FM5044,FIBERMAX FM5045, FIBERMAX FM5013, FIBERMAX FM5015, FIBERMAX FM5017 orFIBERMAX FM5024 and plants with genotypes derived thereof. These aresuitable for introducing the chimeric gene disclosed herein.

It is also an object of the invention to provide plant cells, plantparts and plants generated according to the methods of the invention,such as fruits, seeds, embryos, reproductive tissue, meristematicregions, callus tissue, leaves, roots, shoots, flowers, fibers, vasculartissue, gametophytes, sporophytes, pollen and microspores, which arecharacterised in that they comprise a specific modification in thegenome (insertion, replacement and/or deletion) or in that they comprisethe chimeric HDDP gene according to the invention. Gametes, seeds,embryos, either zygotic or somatic, progeny or hybrids of plantscomprising the DNA modification events, which are produced bytraditional breeding methods, are also included within the scope of thepresent invention. Such plants may contain a DNA of interest inserted ator instead of a target sequence or may have a specific DNA sequencedeleted (even single nucleotides), and will only be different from theirprogenitor plants by the presence of this heterologous DNA or DNAsequence or the absence of the specifically deleted sequence postexchange. Alternatively, such plants contain the chimeric HPPD geneaccoding to the invention. It will be clear that the plant cells, plantparts and plants of the invention can be from any plant as listed hereinabove, including all cotton plants.

In particular embodiments the plant cell described herein is anon-propagating plant cell or a plant cell that cannot be regeneratedinto a plant or a plant cell that cannot maintain its life bysynthesizing carbohydrate and protein from the inorganics, such aswater, carbon dioxide, and inorganic salt, through photosynthesis.

Seed is formed by an embryonic plant enclosed together with storednutrients by a seed coat. It is the product of the ripened ovule ofgymnosperm and angiosperm plants, which occurs after fertilization andto a certain extent growth within the mother plant. The seed disclosedherein retain the distinguishing characteristics of the parents, such asseed obtained by selfing or crossing, e.g. hybrid seed (obtained bycrossing two inbred parental lines).

The plant cells and plants described herein such as those obtained bythe methods described herein may be further used in breeding procedureswell known in the art, such as crossing, selfing, and backcrossing.Breeding programs may involve crossing to generate an F1 (first filial)generation, followed by several generations of selfing (generating F2,F3, etc.). The breeding program may also involve backcrossing (BC)steps, whereby the offspring is backcrossed to one of the parentallines, termed the recurrent parent.

“Introgressing” means the integration of a gene in a plant's genome bynatural means, i. e. by crossing a plant comprising the chimeric genedescribed herein with a plant not comprising said chimeric gene. Theoffspring can be selected for those comprising the chimeric gene.

Accordingly, also disclosed herein is a method for producing plantscomprising the introduced trait (i.e. the genomic moidification or HPPDgene according to the invention) comprising the step of crossing theplant disclosed herein with another plant or with itself and selectingfor offspring comprising the introduced trait.

The plant cells and plants described herein and/or obtained by themethods disclosed herein may also be further used in subsequenttransformation procedures, e. g. to introduce a further chimeric gene.

The plants and seeds according to the invention may be further treatedwith a chemical compound, such as a chemical compound selected from thefollowing lists:

Fruits/Vegetables:

Herbicides: Atrazine, Bromacil, Butafenacil, Diuron, Fluazifop,Glufosinate, Glyphosate, Halosulfuron, Halosulfuron-methyl, Indaziflam,Linuron, Metribuzin, Paraquat, Propyz amide, Sethoxydim, Simazine,Trifluralin.Insecticides: Abamectin, Acequinocyl, Acetamiprid, Aldicarb,Azadirachtin, Benfuracarb, Bifenazate, Buprofezin, Carbaryl, Carbofuran,Chlorantraniliprole (Rynaxypyr), Chlorpyrifos, Chromafenozide,Clothianidin, Cyanopyrafen, Cyantraniliprole (Cyazypyr), Cyflumetofen,beta-Cyfluthrin, gamma-Cyhalothrin, lambda-Cyhalothrin,alpha-Cypermethrin, Deltamethrin, Diafenthiuron, Dinotefuran,Emamectin-benzoate, Esfenvalerate, Fenamiphos, Fenbutatin-oxid,Flonicamid, Fluacrypyrim, Flubendiamide, Fluensulfone, Fluopyram,Flupyradifurone, Imicyafos, Imidacloprid, Indoxacarb, Metaflumizone,Methiocarb, Methoxyfenozide, Novaluron, Pymetrozine, Pyrifluquinazon,Pyriproxifen, Spinetoram, Spinosad, Spirodiclofen, Spiromesifen,Spirotetramat, Sulfoxaflor, Thiacloprid, Thiamethoxam, Thiodicarb,Triflumuron,1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide,1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-1H-tetrazol-1-yl]methyl}-1H-pyrazole-5-carboxamide,1-{2-fluoro-4-methyl-5-[(2,2,2-trifluorethyl)sulfinyl]phenyl}-3-(trifluoromethyl)-1H-1,2,4-triazol-5-amine,(1E)-N-[(6-chloropyridin-3-yl)methyl]-N′-cyano-N-(2,2-difluoroethyl)ethanimidamide,Bacillus firmus, Bacillus firmus strain I-1582, Bacillus thuriengiensis,Bacillus subtilis, Bacillus subtilis strain GB03, Bacillus subtilisstrain QST 713, Metarhizium anisopliae, Metarhizium anisopliae strainF52.Fruits/Vegetables Fungicides: Ametoctradin, Amisulbrom, Azoxystrobin,Benthiavalicarb,N-[9-(dichloromethylene)-1,2,3,4-tetrahydro-1,4-methanonaphthalen-5-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide (Benzovindiflupyr, Benzodiflupyr), Bixafen, Boscalid,Cap tan, Carbendazim, Chlorothalonil, Copper, Cyazofamid, Cyflufenamid,Cymoxanil, Cyproconazole, Cyprodinil, Difenoconazole, Dimetomorph,Dithianon, Epoxiconazole, Famoxadone, Fenamidone, Fenhexamid,Fenpyrazamine, Fluazinam, Fludioxonil, Fluopicolide, Fluopyram,Fluoxastrobin, Fluquinconazole, Fluthianil, Fluxapyroxad, Folpet,Fosetyl, Iprodione, Iprovalicarb, Isopyrazam, Isotianil,Kresoxim-methyl, Mancozeb, Mandipropamid, Meptyldinocap,Metalaxyl/Mefenoxam, Metiram, Metrafenone, Myclobutanil, Penconazole,Penflufen, Penthiopyrad, Phosphonic acid (H3PO3), Picoxystrobin,Propamocarb, Propiconazole, Propineb, Proquinazid, Prothioconazole,Pyraclostrobin, Pyrimethanil, Pyriofenone,1-(4-{4-[(5R)-5-(2,6-difluorophenyl)-4,5-dihydro-1,2-oxazol-3-yl]-1,3-thiazol-2-yl}piperidin-1-yl)-2-[5-methyl-3-(trifluoromethyl)-1H-pyrazol-1-yl]ethanone,Quinoxyfen, Sedaxane, Spiroxamine, Sulphur, Tebuconazole,Thiophanate-methyl, Thiram, Triadimenol, Trifloxystrobin.

Cereals:

Herbicides: 2,4-D, Amidosulfuron, Beflubutamid, Bentazon, Bifenox,Bromoxynil, Carfentrazone-ethyl, Chlorotoluron, Chlorsulfuron,Cinidon-ethyl, Clodinafop-propargyl, Clopyralid, Dicamba, Dichlorprop,Dichlorprop-P, Diclofop-methyl, Diflufenican, Fenoxaprop, Florasulam,Flucarbazone-sodium, Flufenacet, Flupyrsulfuron-methyl-sodium,Fluroxypyr, Flurtamone, Glyphosate, Imazamox, Imazamethabenz,Iodosulfuron, Ioxynil, Isoproturon, Isoxaben, MCPA, MCPB, Mecoprop-P,Mesosulfuron, Metsulfuron, Pendimethalin, Picolinafen, Pinoxaden, Propoxycarbazone, Prosulfocarb, Pyraflufen, Pyrasulfotole, Pyroxsulam,Sulfosulfuron, Thiencarbaz one, Thifensulfuron, Traloxydim, Triallat,Triasulfuron, Tribenuron, Trifluralin, Tritosulfuron (Safener: Mefenpyr,Mefenpyr-diethyl, Cloquintocet).Fungicides: Azoxystrobin,N-[9-(dichloromethylene)-1,2,3,4-tetrahydro-1,4-methanonaphthalen-5-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide(Benzovindiflupyr, Benzodiflupyr), Bixafen, Boscalid, Carbendazim,Carboxin, Chlorothalonil, Cyflufenamid, Cyproconazole, Cyprodinil,Difenoconazole, Dimoxystrobin, Diniconazole, Epoxiconazole, Fenpropidin,Fenpropimorph, Fludioxonil, Fluopyram, Fluoxastrobin, Fluquinconazole,Flusilazole, Fluthianil, Flutriafol, Fluxapyroxad, Imazalil, Ipconazole,Isopyrazam, Kresoxim-methyl, Mefenoxam, Metalaxyl, Metconazole,Metominostrobin, Metrafenone, Myclobutanil, Penflufen, Penthiopyrad,Picoxystrobin, Prochloraz, Propiconazole, Proquinazid, Prothioconazole,Pyraclostrobin, Pyriofenone, Quinoxyfen, Sedaxane, Silthiofam,Spiroxamine, Tebuconazole, Thiabendazole, Thiophanate-methyl,Triadimenol, Trifloxystrobin, Triticonazole, Bacillus firmus, Bacillusfirmus strain I-1582, Bacillus subtilis, Bacillus subtilis strain GB03,Bacillus subtilis strain QST 713.Insecticides: Acetamiprid, Azadirachtin, Benfuracarb, Bifenthrin,Chlorantraniliprole (Rynaxypyr), Chlorpyriphos, Clothianidin,Cyantraniliprole (Cyazypyr), beta-Cyfluthrin, alpha-Cypermethrin,Deltamethrin, Diafenthiuron, Dimethoate, Dinetofuran, Fipronil,Fluensulfone, Fluopyram, Flupyradifurone, Imicyafos, Imidacloprid,Metaflumizone, Metamidophos, Methiocarb, Phorate, Pirimicarb,Pymetrozine, Pyrifluquinaz on, Spirotetramate, Sulfoxaflor, Tefluthrin,Thiacloprid, Thiamethoxam, Thiodicarb, Transfluthim, Triflumuron,1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide,1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-1H-tetrazol-1-yl]methyl}-1H-pyrazole-5-carboxamide,1-{2-fluoro-4-methyl-5-[(2,2,2-trifluorethyl)sulfinyl]phenyl}-3-(trifluoromethyl)-1H-1,2,4-triazol-5-amine,(1E)-N-[(6-chloropyridin-3-yl)methyl]-N′-cyano-N-(2,2-difluoroethyl)ethanimidamide,Bacillus firmus, Bacillus firmus strain I-1582, Bacillus subtilis,Bacillus subtilis strain GB03, Bacillus subtilis strain QST 713,Metarhizium anisopliae F52.

Maize:

Herbicides/Plant Growth Regulators (PGRs): Aclonifen, Atrazine,Alachlor, Bentazon, Bicyclopyrone, Bromoxynil, Acetochlor, Aclonifen,Dicamba, Clopyralid, Dimethenamid-P, Florasulam, Flufenacet, Fluroxypyr,Foramsulfuron, Glufosinate, Glyphosate, Isoxadifen, Isoxaflutole,(S-)Metolachlor, Mesotrione, Nicosulfuron, Pendimethalin, Pethoxamid,Primisulfuron, Pyroxasulfon, Rimsulfuron, Sulcotrione, Tembotrione,Terbuthylazin, Thiencarbaz one, Thifensulfuron-methyl, Topramezone,Saflufenacil (Safener: Isoxadifen-ethyl).Insecticides: Abamectin, Acetamiprid, Azadirachtin, Benfuracarb,Bifenthrin, Carbofuran, Chlorantraniliprole (Rynaxypyr), Chlorpyrifos,Clothianidin, Cyantraniliprole (Cyazypyr), (beta-) Cyfluthrin,lambda-Cyhalothrin, (alpha-)Cypermethrin, Deltamethrin, Dinotefuran,Ethiprole, Fipronil, Flubendiamide, Fluensulfone, Fluopyram,Flupyradifurone, Indoxacarb, Imicyafos, Imidacloprid, Lufenuron,Metaflumiz one, Methiocarb, Pymetrozine, Pyrifluquinazon, Spinetoram,Spinosad, Spiromesifen, Spirotetramate, Sulfoxaflor, Tefluthrin,Terbufos, Thiamethoxam, Thiodicarb, Triflumuron, Tebupirimphos,Thiacloprid,1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide,1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-1H-tetrazol-1-yl]methyl}-1H-pyrazole-5-carboxamide,1-{2-fluoro-4-methyl-5-[(2,2,2-trifluorethyl)sulfinyl]phenyl}-3-(trifluoromethyl)-1H-1,2,4-triazol-5-amine,(1E)-N-[(6-chloropyridin-3-yl)methyl]-N′-cyano-N-(2,2-difluoroethyl)ethanimidamide,Bacillus firmus, Bacillus firmus strain I-1582, Bacillus subtilis,Bacillus subtilis strain GB03, Bacillus subtilis strain QST 713,Metarhizium anisopliae F52.Fungicides: Azoxystrobin,N-[9-(dichloromethylene)-1,2,3,4-tetrahydro-1,4-methanonaphthalen-5-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide(Benzovindiflupyr, Benzodiflupyr), Bixafen, Boscalid, Carbendazim,Chenopodium quinjoa, Cyproconazole, Dimoxystrobin, Epoxiconazole,Fenamidone, Fipronil, Fluopyram, Fluthianil, Fluoxastrobin, Flusilazole,Flutriafol, Fluxapyroxad, Ipconazole, Isopyrazam, Mancozeb, Mefenoxam,Metalaxyl, Metominostrobin, Metconazole, Myclobutanil, Penflufen,Penthiopyrad, Picoxystrobin, Propiconazole, Prothioconazole,Pyraclostrobin, Saponin, Sedaxane, Tebuconazole, Thiram, Triadimenol,Trifloxystrobin, Ziram, Bacillus firmus, Bacillus firmus strain 1-1582,Bacillus subtilis, Bacillus subtilis strain GB03, Bacillus subtilisstrain QST 713, Bacillus pumulis, Bacillus. pumulis strain GB34.

Rice:

Herbicides: Anilofos, Azimsulfuron, Benfuresate, Bensulfuron, Bentazone,Benzobicyclon, Benzofenap, Bispyribac, Bromobutide, Butachlor,Cafenstrole, Carfentrazone-ethyl, Clomazone, Clomeprop Cumyluron,Cyhalofop, Daimuron, Dimepiperate, Esprocarb, Ethoxysulfuron,Fenoxaprop, Fenoxasulfone, Fentrazamide, Flucetosulfuron, Flufenacet,Halosulfuron, Imazosulfuron, Indanofan, Ipfencarbazone, Mefenacet,Mesotrione, Metamifop, Metazosulfuron, Molinate, Naproanilide,Orthosulfamuron, Oxadiargyl, Oxadiazone, Oxaziclomefone, Penoxsulam,Pentoxazone, Pretilachlor, Profoxydim, Propanil, Propyrisulfuron,Pyraclonil, Pyrazolate, Pyrazosulfuron, Pyrazoxyfen, Pyributicarb,Pyriftalid, Pyriminobac-methyl, Pyrimisulfan, Quinclorac, Tefuryltrione,Thenylchlor, Thiobencarb, Triafamone.Insecticides: Acetamiprid, Azadirachtin, Benfuracarb, Buprofezin,Carbofuran, Chlorantraniliprole (Rynaxypyr), Chlorpyriphos,Chromafenozide, Clothianidin, Cyantraniliprole (Cyazypyr), Cypermethrin,Deltamethrin, Diazinon, Dinotefuran, Emamectin-benzoate, Ethiprole,Etofenprox, Fenobucarb, Fipronil, Flubendiamide, Fluensulfone,Fluopyram, Flupyradifurone, Imicyafos, Imidacloprid, Isoprocarb,Metaflumizone, Pymetrozine, Pyrifluquinazon, Spinetoram, Spinosad,Spirotetramate Sulfoxaflor, Thiacloprid, Thiamethoxam,1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide,1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-1H-tetrazol-1-yl]methyl}-1H-pyrazole-5-carboxamide,1-{2-fluoro-4-methyl-5-[(2,2,2-trifluorethyl)sulfinyl]phenyl}-3-(trifluoromethyl)-1H-1,2,4-triazol-5-amine,(1E)-N-[(6-chloropyridin-3-yl)methyl]-N′-cyano-N-(2,2-difluoroethyl)ethanimidamide,Bacillus firmus, Bacillus firmus strain I-1582, Bacillus subtilis,Bacillus subtilis strain GB03, Bacillus subtilis strain QST 713,Metarhizium anisopliae F52.Fungicides: Azoxystrobin,N-[9-(dichloromethylene)-1,2,3,4-tetrahydro-1,4-methanonaphthalen-5-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide(Benzovindiflupyr, Benzodiflupyr), Bixafen, Carbendazim, Carpropamid,Chlorothalonil, Copper-oxychloride, Cyproconazole, Diclocymet,Difenoconazole, Edifenphos, Epoxiconazole, Ferimzone, Fludioxonil,Fluopyram, Fluoxastrobin, Flusilazole, Fluthianil, Flutolanil,Fluxapyroxad, Furametpyr, Gentamycin, Hexaconazole, Hymexazol,Ipconazole, Iprobenfos (IBP), Iprodione, Isoprothiolane, Isotianil,Kasugamycin, Mancozeb, Mefenoxam, Metalaxyl, Metominostrobin,Myclobutanil, Orysastrobin, Pencycuron, Penflufen, Phthalide,Probenazole, Prochloraz, Propamocarb, Propiconazole, Propineb,Prothioconazole, Pyroquilon, Sedaxane, Simconazole, Streptomycin,Sulphur, Tebuconazole, Thifluzamide, Thiophanate-methyl, Thiram,Tiadinil, Tricyclazole, Trifloxystrobin, Validamycin, Bacillus firmus,Bacillus firmus strain 1-1582, Bacillus subtilis, Bacillus subtilisstrain GB03, Bacillus subtilis strain QST 713.

Cotton:

Herbicides: Carfentrazone, Clethodim, Diuron, Fluazifop-butyl,Flumioxazin, Fluometuron, Glufosinate, Glyphosate, MSMA, Norflurazon,Oxyfluorfen, Pendimethalin, Prometryn, Pyrithiobac-sodium, Tepraloxydim,Thidiazuron, Trifloxysulfuron, Trifluralin.

Insecticides: Abamectin, Acephate, Acetamiprid, Aldicarb, Azadirachtin,Bifenthrin, Chlorantraniliprole (Rynaxypyr), Chlorpyrifos, Clothianidin,Cyantraniliprole (Cyazypyr), (beta-)Cyfluthrin, gamma-Cyhalothrin,lambda-Cyhalothrin, Cypermethrin, Deltamethrin, Diafenthiuron,Dinotefuran, Emamectin-benzoate, Flonicamid, Flubendiamide,Fluensulfone, Fluopyram, Flupyradifurone, Imicyafos, Imidacloprid,Indoxacarb, Metaflumizone, Pymetrozine, Pyridalyl, Pyrifluquinazon,Spinetoram, Spinosad, Spiromesifen, Spirotetramat, Sulfoxaflor,Thiacloprid, Thiamethoxam, Thiodicarb, Triflumuron,1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide,1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-1H-tetrazol-1-yl]methyl}-1H-pyrazole-5-carboxamide,1-{2-fluoro-4-methyl-5-[(2,2,2-trifluorethyl)sulfinyl]phenyl}-3-(trifluoromethyl)-1H-1,2,4-triazol-5-amine,(1E)-N-[(6-chloropyridin-3-yl)methyl]-N′-cyano-N-(2,2-difluoroethyl)ethanimidamide,Bacillus firmus, Bacillus firmus strain I-1582, Bacillus subtilis,Bacillus subtilis strain GB03, Bacillus subtilis strain QST 713,Metarhizium anisopliae F52.Fungicides: Azoxystrobin,N-[9-(dichloromethylene)-1,2,3,4-tetrahydro-1,4-methanonaphthalen-5-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide(Benzovindiflupyr, Benzodiflupyr), Bixafen, Boscalid, Carbendazim,Chlorothalonil, Copper, Cyproconazole, Difenoconazole, Dimoxystrobin,Epoxiconazole, Fenamidone, Fluazinam, Fluopyram, Fluoxastrobin,Fluxapyroxad, Ipconazole, Iprodione, Isopyrazam, Isotianil, Mancozeb,Maneb, Mefenoxam, Metalaxyl, Metominostrobin, Pencycuron, Penflufen,Penthiopyrad, Picoxystrobin, Propineb, Prothioconazole, Pyraclostrobin,Quintozene, Sedaxane, Tebuconazole, Tetraconazole, Thiophanate-methyl,Triadimenol, Trifloxystrobin, Bacillus firmus, Bacillus firmus strain1-1582, Bacillus subtilis, Bacillus subtilis strain GB03, Bacillussubtilis strain QST 713.

Soybean:

Herbicides: Alachlor, Bentaz one, Chlorimuron-Ethyl, Cloransulam-Methyl,Fenoxaprop, Fluazifop, Fomesafen, Glufosinate, Glyphosate, Imazamox,Imazaquin, Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin,Tepraloxydim, Trifluralin.Insecticides: Acetamiprid, Azadirachtin, Chlorantraniliprole(Rynaxypyr), Clothianidin, Cyantraniliprole (Cyazypyr), (beta-)Cyfluthrin, gamma-Cyhalothrin, lambda-Cyhalothrin, Deltamethrin,Dinotefuran, Emamectin-benzoate, Ethiprole, Fipronil, Flonicamid,Flubendiamide, Fluensulfone, Fluopyram, Flupyradifurone, Imicyafos,Imidacloprid, Metaflumizone, Methomyl, Pyrifluquinaz on, Pymetrozine,Spinetoram, Spinosad, Spirodiclofen, Spiromesifen, Spirotetramat,Sulfoxaflor, Thiacloprid, Thiamethoxam, Thiodicarb, Triflumuron,1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide,1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-1H-tetrazol-1-yl]methyl}-1H-pyrazole-5-carboxamide,1-{2-fluoro-4-methyl-5-[(2,2,2-trifluorethyl)sulfinyl]phenyl}-3-(trifluoromethyl)-1H-1,2,4-triazol-5-amine,(1E)-N-[(6-chloropyridin-3-yl)methyl]-N′-cyano-N-(2,2-difluoroethyl)ethanimidamide,Bacillus firmus, Bacillus firmus strain I-1582, Bacillus subtilis,Bacillus subtilis strain GB03, Bacillus subtilis strain QST 713,Metarhizium anisopliae F52, Rhizobia.Fungicides: Azoxystrobin,N-[9-(dichloromethylene)-1,2,3,4-tetrahydro-1,4-methanonaphthalen-5-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide(Benzovindiflupyr, Benzodiflupyr), Bixafen, Boscalid, Carbendazim,Carboxin, Chenopodium quinoa saponins, Chlorothalonil, Copper,Cyproconazole, Difenoconazole, Dimoxystrobin, Epoxiconazole, Fluazinam,Fludioxonil, Fluopicolide, Fluopyram, Fluoxastrobin, Fluquinconazole,Flutriafol, Fluthianil, Fluxapyroxad, Ipconazole, Isopyraz am,Iprodione, Is otianil, Mancozeb, Maneb, Mefenoxam, Metalaxyl,Metconazole, Metominostrobin, Myclobutanil, Penflufen, Penthiopyrad,Picoxystrobin, Propiconazole, Propineb, Prothioconazole, Pyraclostrobin,Sedaxane, Tebuconazole, Tetraconazole, Thiophanate-methyl, Thiram,Triadimenol, Trifloxystrobin, Bacillus subtilis, Bacillus subtilisstrain GB03, Bacillus subtilis strain QST 713, Bacillus pumilis,Bacillus pumilis GB34.

Sugarbeet:

Herbicides: Chloridazon, Clopyralid, Cycloxydim, Desmedipham,Ethofumesate, Fluazifop, Lenacil, Metamitron, Phenmedipham, Quinmerac,Quizalofop, Tepraloxydim, Triallate, Triflusulfuron. Insecticides:Acetamiprid, Aldicarb, Az adirachtin, Clothianidin, Dinetofuran,Deltamethrin, Carbofuran, Chlorantraniliprole (Rynaxypyr),Cyantraniliprole (Cyazypyr), (beta-)Cyfluthrin, gamma-Cyhalothrin,lambda-Cyhalothrin, Cypermethrin, Fipronil, Fluensulfone, Fluopyram,Flupyradifurone, Imicyafos, Imidacloprid, Metaflumizone, Methiocarb,Pymetrozine, Pyrifluquinaz on, Spirotetramate, Sulfoxaflor, Tefluthrin,Thiacloprid, Thiamethoxam,1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide,1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-1H-tetrazol-1-yl]methyl}-1H-pyrazole-5-carboxamide,1-{2-fluoro-4-methyl-5-[(2,2,2-trifluorethyl)sulfinyl]phenyl}-3-(trifluoromethyl)-1H-1,2,4-triazol-5-amine,(1E)-N-[(6-chloropyridin-3-yl)methyl]-N′-cyano-N-(2,2-difluoroethyl)ethanimidamide,Bacillus firmus, Bacillus firmus strain I-1582, Bacillus subtilis,Bacillus subtilis strain GB03, Bacillus subtilis strain QST 713,Metarhizium anisopliae F52.Fungicides: Azoxystrobin,N-[9-(dichloromethylene)-1,2,3,4-tetrahydro-1,4-methanonaphthalen-5-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide(Benzovindiflupyr, Benzodiflupyr), Bixafen, Carbendazim, Chlorothalonil,Copper, Cyproconazole, Difenoconazole, Epoxiconazole, Fefenoxam,Fenpropidin, Fenpropimorph, Fluopyram, Flusilazole, Fluthianil,Flutriafol, Fluxapyroxad, Hymexazol, Ipconazole, Isopyrazam,Kresoxim-methyl, Mancozeb, Maneb, Metalaxyl, Myclobutanil, Penflufen,Prochloraz, Propiconazole, Prothioconazole, Tebuconazole,Pyraclostrobin, Quinoxyfen, Sedaxane, Sulphur, Tetraconazole,Thiophanate-methyl, Thiram, Trifloxystrobin, Bacillus firmus, Bacillusfirmus strain I-1582, Bacillus subtilis, Bacillus subtilis strain GB03,Bacillus subtilis strain QST 713.

Canola:

Herbicides: Clethodim, Clopyralid, Diclofop, Ethametsulfuron, Fluazifop,Glufosinate, Glyphosate, Metazachlor, Quinmerac, Quizalofop,Tepraloxydim, Trifluralin.

Fungicides/PGRs: Azoxystrobin,N-[9-(dichloromethylene)-1,2,3,4-tetrahydro-1,4-methanonaphthalen-5-yl]-3-(difluoromethyl)-1-methyl-1H-pyrazole-4-carboxamide(Benzovindiflupyr, Benzodiflupyr), Bixafen, Boscalid, Carbendazim,Carboxin, Chlormequat-chloride, Coniothryrium minitans, Cyproconazole,Cyprodinil, Difenoconazole, Dimethomorph, Dimoxystrobin, Epoxiconazole,Famoxadone, Fluazinam, Fludioxonil, Fluopicolide, Fluopyram,Fluoxastrobin, Fluquinconazole, Flusilazole, Fluthianil, Flutriafol,Fluxapyroxad, Iprodione, Isopyrazam, Mefenoxam, Mepiquat-chloride,Metalaxyl, Metconazole, Metominostrobin, Paclobutrazole, Penflufen,Penthiopyrad, Picoxystrobin, Prochloraz, Prothioconazole,Pyraclostrobin, Sedaxane, Tebuconazole, Tetraconazole,Thiophanate-methyl, Thiram, Triadimenol, Trifloxystrobin, Bacillusfirmus, Bacillus firmus strain 1-1582, Bacillus subtilis, Bacillussubtilis strain GB03, Bacillus subtilis strain QST 713, Bacilluspumulis, Bacillus. pumulis strain GB34.Insecticides: Acetamiprid, Aldicarb, Azadirachtin, Carbofuran,Chlorantraniliprole (Rynaxypyr), Clothianidin, Cyantraniliprole(Cyazypyr), (beta-)Cyfluthrin, gamma-Cyhalothrin, lambda-Cyhalothrin,Cypermethrin, Deltamethrin, Dimethoate, Dinetofuran, Ethiprole,Flonicamid, Flubendiamide, Fluensulfone, Fluopyram, Flupyradifurone,tau-Fluvalinate, Imicyafos, Imidacloprid, Metaflumizone, Methiocarb,Pymetrozine, Pyrifluquinazon, Spinetoram, Spinosad, Spirotetramate,Sulfoxaflor, Thiacloprid, Thiamethoxam,1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide,1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-1H-tetrazol-1-yl]methyl}-1H-pyrazole-5-carboxamide,1-{2-fluoro-4-methyl-5-[(2,2,2-trifluorethyl)sulfinyl]phenyl}-3-(trifluoromethyl)-1H-1,2,4-triazol-5-amine,(1E)-N-[(6-chloropyridin-3-yl)methyl]-N′-cyano-N-(2,2-difluoroethyl)ethanimidamide,Bacillus firmus, Bacillus firmus strain I-1582, Bacillus subtilis,Bacillus subtilis strain GB03, Bacillus subtilis strain QST 713,Metarhizium anisopliae F52.

Further disclosed herein is a fiber or oil obtained from the cottonplant or seed disclosed herein comprising an HPPD gene according to theinvention. Further disclosed herein is yarn, fabric or filler comprisingthe fiber disclosed herein as well a meal comprising at least a part ofthe seed of the invention.

In another aspect, the present application discloses a method forobtaining a cotton plant or plant cell tolerant to a field dose of atleast 1× of at least one HPPD inhibitor, comprising introducing achimeric gene comprising (a) a nucleic acid sequence encoding a proteinhaving HPPD activity, wherein said protein has a tryptophan at aposition corresponding to position 336 of SEQ ID NO: 19, wherein saidprotein provides to said plant tolerance to a field dose of at least 1×of at least one HPPD inhibitor, operably linked to (b) a plantexpressible promoter and optionally (c) a translational termination andpolyadenylation region into a cotton plant cell.

The method may further comprise growing a plant from the plant cell.

Also disclosed herein is a method for controlling weeds in the vicinityof a cotton plant or on a field of cotton plants according to theinvention, comprising applying at least one HPPD inhibitor to thevicinity of said cotton plant or to a cotton plant field in a field doseof at least 1×.

In a particular embodiment, the at least one HPPD inhibitor is appliedpre-emergence and the same or another at least one HPPD inhibitor isapplied post-emergence. For example, the pant or field of plants may betreated with a pre-emergence dose of at least 1× of e.g. IFT followed bya post-emergence dose of at least 1× of the same or another HPPDinhibitor, e.g. MST or TBT.

The term “weed”, as used herein, refers to undesired vegetation on e.g.a field, or to plants, other then the intentionally planted plants,which grow unwantedly between the plants of interest and may inhibitgrowth and development of said plants of interest. The term “controllingweeds” thus includes inhibition of weed growth and killing of weeds.

The vicinity of a plant of interest includes the area around it, inparticular the area covered by the roots of a plant through all stagesof growth.

At least one HPPD inhibitor is applied to the cotton field or to thevicinity of a cotton plant by spraying a solution comprising said HPPDinhibitor(s) on a cotton field or in the vicinity of a cotton plant suchthat a concentration of said at least one HPPD inhibitor according tothe desired field dose is obtained.

In a particular embodiment, the at least one HPPD inhibitor is appliedpre-emergence and the same or another at least one HPPD inhibitor isapplied post-emergence. For example, the pant or field of plants may betreated with a pre-emergence dose of at least 1× of IFT and apost-emergence dose of at least 1× of the same or another HPPDinhibitor, such as MST.

In one example, at least one HPPD inhibitor is applied in a dose whichis toxic for said weeds. Such a dose is usually 1× but may in individualcases be higher such as 1,5×, 2×, 4× or even more. As already indicatedabove, dose rates corresponding to 1× may vary depending on manyfactors. In any case, the dose rate for a given situation may beincreased.

As used herein, the amount or concentration of an HPPD inhibitor “toxicfor a weed” is interchangeably used with the term “effective amount” or“effective concentration.” An “effective amount” and “effectiveconcentration” is an amount or concentration that is sufficient to killor inhibit the growth of a weed, but that said amount does not kill orinhibit as severely the growth of the cotton plants, plant tissues,plant cells, and seeds disclosed herein. Typically, the effective amountof at least one HPPD inhibitor is an amount that is routinely used inagricultural production systems to kill weeds of interest, expressed asa “field dose”. In this regard, for cotton, a field dose of 1× maydiffer depending on the HPPD inhibitor chosen as has been exemplifiedabove.

In one example, said at least one HPPD inhibitor is applied in a fielddose of at least 1.5×.

In a further example said at least one HPPD inhibitor is applied in afield dose of at least 2×.

In yet another example, said at least one HPPD inhibitor is applied in afield dose of at least 3× or at least 4×.

Said at least one HPPD inhibitor may be applied post-emergence but alsopre-emergence, or both.

Also disclosed herein is a method of producing a cotton seed or a cottonfiber comprising a chimeric gene comprising (a) a nucleic acid sequenceencoding a protein having HPPD activity, wherein said protein has atryptophan at a position corresponding to position 336 of SEQ ID NO: 19,wherein said protein provides to a cotton plant growing from said seedtolerance to a field dose of at least 1× of at least one HPPD inhibitor,operably linked to (b) a plant expressible promoter; and optionally (c)an translational termination and polyadenylation region, the methodcomprising providing the plant disclosed herein or the plant obtained bythe method described herein above, wherein said plant produces said seedand said chimeric gene is comprised in said seed; and isolating saidseed or said fiber from said cotton plant. In one example, said proteinhaving HPPD activity has at least 95% sequence identity to SEQ ID NO:21, comprises SEQ ID NO: 21 or consists of SEQ ID NO: 19. Other suitableexamples for the protein having HPPD function are disclosed elsewhere inthis description.

The present application also discloses a method for producing fabriccomprising processing the fiber disclosed herein.

Further disclosed is the use of a chimeric gene as disclosed herein,such as a chimeric gene comprising (a) a nucleic acid sequence encodinga protein having HPPD activity, wherein said protein has a tryptophan ata position corresponding to position 336 of SEQ ID NO: 19, wherein saidprotein provides to a plant tolerance to a field dose of at least 1× ofat least one HPPD inhibitor, operably linked to (b) a plant expressiblepromoter and optionally (c) a translational termination andpolyadenylation region for (i) obtaining tolerance to a field dose of atleast 1× of at least one HPPD inhibitor in a cotton plant, or (ii)producing a cotton plant tolerant to a field dose of at least 1× of atleast one HPPD inhibitor.

Also disclosed herein is the use of the cotton fiber disclosed hereinfor producing yarn, fabric or filler material.

Further disclosed herein is the use of the seed disclosed herein, suchas the cotton seed, for producing seed oil or meal.

A chimeric gene is an artificial gene constructed by operably linkingfragments of unrelated genes or other nucleic acid sequences. In otherwords “chimeric gene” denotes a gene which is not normally found in aplant species or refers to any gene in which the promoter or one or moreother regulatory regions of the gene are not associated in nature with apart or all of the transcribed nucleic acid, i. e. are heterologous withrespect to the transcribed nucleic acid. More particularly, a chimericgene is an artificial, i. e. non-naturally occurring, gene produced byan operable linkage of a nucleic acid sequence to be transcribed.

The term “heterologous” refers to the relationship between two or morenucleic acid or protein sequences that are derived from differentsources. For example, a promoter is heterologous with respect to anoperably linked nucleic acid sequence, such as a coding sequence, ifsuch a combination is not normally found in nature. In addition, aparticular sequence may be “heterologous” with respect to a cell ororganism into which it is inserted (i.e. does not naturally occur inthat particular cell or organism). For example, the chimeric genedisclosed herein is a heterologous nucleic acid.

The expression “operably linked” means that said elements of thechimeric gene are linked to one another in such a way that theirfunction is coordinated and allows expression of the coding sequence,i.e. they are functionally linked. By way of example, a promoter isfunctionally linked to another nucleotide sequence when it is capable ofensuring transcription and ultimately expression of said othernucleotide sequence. Two proteins encoding nucleotide sequences, e.g. atransit peptide encoding nucleic acid sequence and a nucleic acidsequence encoding a protein having HPPD activity, are functionally oroperably linked to each other if they are connected in such a way that afusion protein of first and second protein or polypeptide can be formed.

As used herein “comprising” is to be interpreted as specifying thepresence of the stated features, integers, steps or components asreferred to, but does not preclude the presence or addition of one ormore features, integers, steps or components, or groups thereof. Thus,e.g., a nucleic acid or protein comprising a sequence of nucleotides oramino acids, may comprise more nucleotides or amino acids than theactually cited ones, i.e., be embedded in a larger nucleic acid orprotein. A chimeric gene comprising a DNA region which is functionallyor structurally defined may comprise additional DNA regions etc.

As used herein, “plant part” includes any plant organ or plant tissue,including but not limited to fruits, seeds, embryos, meristematicregions, callus tissue, leaves, roots, shoots, flowers, gametophytes,sporophytes, pollen, and microspores.

For the purpose of this invention, the “sequence identity” of tworelated nucleotide or amino acid sequences, expressed as a percentage,refers to the number of positions in the two optimally aligned sequenceswhich have identical residues (×100) divided by the number of positionscompared. A gap, i.e. a position in an alignment where a residue ispresent in one sequence but not in the other, is regarded as a positionwith non-identical residues. The optimal alignment of the two sequencesis performed by the Needleman and Wunsch algorithm (Needleman and Wunsch1970). The computer-assisted sequence alignment above, can beconveniently performed using standard software program such as GAP whichis part of the Wisconsin Package Version 10.1 (Genetics Computer Group,Madison, Wis., USA) using the default scoring matrix with a gap creationpenalty of 50 and a gap extension penalty of 3.

Nucleic acids can be DNA or RNA, single- or double-stranded. Nucleicacids can be synthesized chemically or produced by biological expressionin vitro or even in vivo. Nucleic acids can be chemically synthesizedusing appropriately protected ribonucleoside phosphoramidites and aconventional DNA/RNA synthesizer. Suppliers of RNA synthesis reagentsare Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo.,USA), Pierce Chemical (part of Perbio Science, Rockford, Ill., USA),Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA), andCruachem (Glasgow, UK). In connection with the chimeric gene of thepresent disclosure, DNA includes cDNA and genomic DNA.

The terms “protein” or “polypeptide” as used herein describe a group ofmolecules consisting of more than 30 amino acids, whereas the term“peptide” describes molecules consisting of up to 30 amino acids.Proteins and peptides may further form dimers, trimers and higheroligomers, i.e. consisting of more than one (poly)peptide molecule.Protein or peptide molecules forming such dimers, trimers etc. may beidentical or non-identical. The corresponding higher order structuresare, consequently, termed homo- or heterodimers, homo- or heterotrimersetc. The terms “protein” and “peptide” also refer to naturally modifiedproteins or peptides wherein the modification is effected e.g. byglycosylation, acetylation, phosphorylation and the like. Suchmodifications are well known in the art.

It will be clear that whenever nucleotide sequences of RNA molecules aredefined by reference to nucleotide sequence of corresponding DNAmolecules, the thymine (T) in the nucleotide sequence should be replacedby uracil (U). Whether reference is made to RNA or DNA molecules will beclear from the context of the application.

The following non-limiting Examples describe the use of a re-designedmeganuclease to introduce a DNA of interest in close proximity to theGHB119 elite event and the generation of cotton plants comprising anHPPD gene conferring tolerance to a field dose of at least 1× of atleast one HPPD inhibitor.

Unless stated otherwise in the Examples, all recombinant DNA techniquesare carried out according to standard protocols as described in Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 ofAusubel et al. (1994) Current Protocols in Molecular Biology, CurrentProtocols, USA. Standard materials and methods for plant molecular workare described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy,jointly published by BIOS Scientific Publications Ltd (UK) and BlackwellScientific Publications, UK. Other references for standard molecularbiology techniques include Sambrook and Russell (2001) MolecularCloning: A Laboratory Manual, Third Edition, Cold Spring HarborLaboratory Press, NY, Volumes I and II of Brown (1998) Molecular BiologyLabFax, Second Edition, Academic Press (UK). Standard materials andmethods for polymerase chain reactions can be found in Dieffenbach andDveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring HarborLaboratory Press, and in McPherson at al. (2000) PCR—Basics: FromBackground to Bench, First Edition, Springer Verlag, Germany.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference, in their entireties, for all purposes.

Throughout the description and Examples, reference is made to thefollowing sequences:

SEQ ID NO. 1: Recognition sequence of the BAY-5/6 meganuclease (sense)

SEQ ID NO. 2: Recognition sequence of the BAY-5/6 meganuclease (reversecomplement of SEQ ID NO. 1)

SEQ ID NO. 3: 3′ flanking sequence of GHB119 containing the BAY-5/6recognition site

SEQ ID NO. 4: 5′ flanking sequence of GHB119

SEQ ID NO. 5: meganuclease expression vector pCV193

SEQ ID NO. 6: Amino acid sequence of the COT-5/6 single chainmeganuclease

SEQ ID NO. 7: Repair DNA vector pCV211

SEQ ID NO. 8: Amino acid sequence of Pf-HPPD (W336)

SEQ ID NO. 9: Amino acid sequence of 2mEPSPS

SEQ ID NO. 10: Nucleotide sequence of Cry2A probe

SEQ ID NO. 11: Nucleotide sequence of HPPD probe

SEQ ID NO. 12: Nucleotide sequence of 2mEPSPS probe

SEQ ID NO. 13: PCR primer IB527

SEQ ID NO. 14: PCR primer IB616

SEQ ID NO. 15: PCR primer IB617

SEQ ID NO. 16: Amino acid sequence of I-CreI

SEQ ID NO. 17: PCR primer IB589

SEQ ID NO. 18: PCR primer VDS406

SEQ ID NO. 19: Amino acid sequence of the wild type (wt) Pseudomonasfluorescens HPPD protein

SEQ ID NO. 20: Nucleic acid sequence of T-DNA of vector pTIF16,comprising a chimeric gene according to the invention

SEQ ID NO. 21: Amino acid sequence encoding the Pseudomonas fluorescensHPPD protein wherein the glycine at amino acid position 336 has beenreplaced by a tryptophan

SEQ ID NO: 22: Amino acid sequence of the 2mEPSPS protein of Z. mays

SEQ ID NO. 23: Nucleic acid sequence of T-DNA of vector pTSIH09,comprising a chimeric gene according to the invention

The sequence listing contained in the file named “BCS11-2012-WO_ST25”,which is 105 kilobytes (size as measured in Microsoft Windows®),contains 23 sequences SEQ ID NO. 1 through SEQ ID NO: 23, is filedherewith by electronic submission and is incorporated by referenceherein.

EXAMPLES

All re-designed meganucleases and their expression vectors describedherein have been designed by Precision BioSciences Inc., 104 T. W.Alexander Drive, Research Triangle Park, N. C.27713.

Example 1 Vector Construction

Using standard recombinant DNA techniques, the following DNA vector wasconstructed comprising the following operably linked elements(schematically depicted in FIG. 3):

Repair DNA vector pCV211 (SEQ ID NO: 7)

-   -   Nt 2252-4310: GHB119 upstream COT-5/6 recognition site:        3′flanking genomic DNA of event GHB119 upstream of the COT-5/6        recognition site.    -   4354-4771: P35S2c: P35S2 promoter sequence.    -   Nt 4772-4840: 5′cab22L: Sequence including the leader sequence        of the chlorophyl a/b binding protein gene of Petunia hybrida        (Harpster et al., 1988).    -   Nt 4842-5213: TPotpY-1Pa: coding sequence of an optimized        transit peptide derivative (position 55 changed into Tyr),        containing sequence of the RuBisCO small subunit genes of Zea        mays (corn) and Helianthus annuus (sunflower) (Lebrun et al.,        1996), adapted for cotton codon usage.    -   Nt 5214-6290 (incl stop): hppdPfW336-1Pa: coding sequence of the        4-hydroxyphenylpyruvate dioxygenase gene of Pseudomonas        fluorescens strain A32 modified by the replacement of the amino        acid Glycine 336 with a Tryptophane (Boudec et al., 1999),        adapted to cotton codon usage.    -   Nt 6316-6976: 3′histonAt: sequence including the 3′ untranslated        region of the histone H4 gene of Arabidopsis thaliana (Chaboute        et al., 1987).    -   Nt 8026-8488: intron1 h3At: first intron of gene II of the        histone H3.III variant of Arabidopsis thaliana (Chaubet et al.,        1992).    -   Nt 8495-8866: TPotpC: coding sequence of the optimized transit        peptide, containing sequence of the RuBisCO small subunit genes        of Zea mays (corn) and Helianthus annuus (sunflower) (Lebrun et        al., 1996).    -   Nt 8867-10204: 2mepsps: coding sequence of the double-mutant        5-enol-pyruvylshikimate-3-phosphate synthase gene of Zea mays        (corn) (Lebrun et al., 1997).    -   Nt 10228-10888: 3′histonAt: sequence including the 3′        untranslated region of the histone H4 gene of Arabidopsis        thaliana (Chabouté et al., 1987).    -   Nt 10971-12521: FGD GHB119: downstream COT-5/6 recognition site:        3′flanking genomic DNA of event GHB119 downstream of COT-5/6        recognition site.

COT-5/6 meganuclease expression vector pCV193 (SEQ ID NO: 5)

-   -   Nt 2241-2599: P35S2c (fragment): The P35S2c promoter fragment.    -   Nt 2602-3083: P35S2c: P35S promoter sequence.    -   Nt 3091-4169: COT-5/6-SC: COT-5/6 single chain meganuclease        encoding DNA region.    -   Nt 4173-4433: 3′nos: sequence including the 3′ untranslated        region of the nopaline synthase gene from the T-DNA of pTiT37        (Depicker et al., 1982).

Example 2 Media and Buffers

Media and buffers used during the embryonic callus generation andtransformation as described below in examples 3 and 4:

Co-cultivation substrate: M100 with ½ concentration MS salts, +100 μMAS+100 mg/L L-cysteïne (L-cysteïne has always to be freshly prepared andadded after autoclavation), pH 5.2M100 substrate: MS salts, B5 vitamins, MES 0.5 g/L, MgCl₂.6H₂O 0.94 g/L,gelrite 2 g/L, glucose 30 g/L, pH 5.8100Q substrate: M100 substrate+0.2 M mannitol+0.2 M sorbitol, pH5.8M104 substrate: =M100 substrate+1 g/L KNO₃, pH 5.8M700 substrate: Stewarts salts+vitamins, MgCl₂.6H₂O 0.47 g/L, gelrite 1g/L, plant agar 2.25 g/L, sucrose 20 g/L, pH 6.8M702 substrate: Stewarts salts+vitamins, MgCl₂.6H₂O 0.71 g/L, gelrite1.5 g/L, plant agar 5 g/L, sucrose 5 g/L, pH 6.8AC: active carbon 2 g/LAS: acetosyringone

Example 3 Generation of Friable Embryogenic Callus

Cotton seeds from Coker 312 were germinated on solid germination mediumM100 without hormones for 7-10 days in the dark at 26-28° C. Next,induction of embryogenic callus was performed by incubating hypocotylexplants from the seedlings on solid M100 medium (without hormones).After about 2 months when the wound callus at the cut surface of thehypocotyls starts to show fast proliferation, the further subculture forenrichment and maintenance of embryogenic callus is done on solid M100medium with active carbon (2 g/L). Induction and maintenance ofembryogenic callus occurs under dim light conditions (intensity: 1 to 7μmol m⁻² sec⁻¹; photoperiod: 16H light/8H dark) at 26-28° C.(Essentially as described in U.S. provisional application 61/493,579 andEP11004570.5, herein incorporated by reference, in particular pagespages 31-35, examples 2-5).

Example 4 Transformation of Cotton Embryogenic Callus by ParticleBombardment

The following procedure was followed to transform cotton embryogeniccallus using particle bombardment

-   -   Friable cotton embryogenic callus (EC) of example 3 of a target        line in which to introduce a DSB induced targeted modification,        is collected 2 to 3 weeks after subculture and plated in a thin        layer by means of a Büchnerfilter on top of a filter paper on        100Q substrate with 0.2M mannitol and 0.2M sorbitol for ˜4 to ˜6        hours prior to bombardment.    -   After preplasmolysis on 100Q substrate for ˜4 to ˜6 hours, the        EC is bombarded with 0.5 pmol pCV211+0.5 pmol pCV193 or 0.75        pmol pCV211+0.5 pmol pCV193 or 0.75 pmol pCV211+0.75 pmol        pCV193.    -   Bombardment conditions:        -   diameter gold particles: 0.3-3 μm        -   rupture disc: 1350 psi        -   distance to target tissue: 9 cm        -   chamber vacuum ˜27 (in Hg)        -   BioRAD PPS_(—)1000/He Biolistic Particle delivering system    -   After bombardment, the filters are transferred onto M100        substrate with 0.2 M mannitol or M100 substrate without        selective agent.    -   After 1 to 4 days on non-selective substrate under dimlight        conditions at 26-28° C., the filters are transferred onto        selective M100 substrate with 1 mM glyphosate.    -   After about 2 to 3 weeks, proliferating calli are selected from        the filters and further subcultured as small piles onto        selective M100 substrate with 1 mM glyphosate. After a        subculture period of ˜6 weeks with ˜3 weekly subculture        intervals, on selective M100 substrate under dimlight conditions        at 26-28° C., transformed EC/somatic embryos can be selected.    -   A molecular screen based on PCR analysis for the identification        of targeted modification events is performed at the level of        transformed EC/somatic embryos.    -   Plant regeneration is initiated from the targeted modification        events by plating EC/somatic embryos on M104 with active carbon        (AC) and the corresponding selective agent under light        conditions (intensity: 40 to 70 μmol m⁻² sec⁻¹; photoperiod: 16H        light/8H dark) at 26-28° C.    -   After about one month individual embryos of about 0.5-1 cm are        transferred on top of a filter paper on M104 with AC and 1 mM        glyphosate.    -   Further well germinating embryos are transferred onto        non-selective germination substrate M702 under light conditions        (intensity: 40 to 70 μmol m⁻² sec⁻¹; photoperiod: 16H light/8H        dark) at 26-28° C.    -   After one to two months the further developing embryos are        transferred onto M700 substrate under light conditions        (intensity: 40 to 70 μmol m⁻² sec⁻¹; photoperiod: 16H light/8H        dark) at 26-28° C. for development into small plantlets.    -   (Essentially as described in U.S. provisional application        61/493,579 and EP11004570.5, herein incorporated by reference,        in particular pages pages 31-35, examples 2-5)

Example 5 Targeted Modification of the Genomic Region in Close Proximityto the GHB119 Transgenic Event Via Homologous Recombination

The pCV211 repair DNA vector and the pCV193 COT-5/6 meganucleaseencoding vector were introduced into embryogenic calli of hemizygousGHB119 plants (described in WO2008/151780) using particle bombardment asdescribed in example 4.

First, transformants were screened for glyphosate resistance, as thisindicates insertion of the repair DNA pCV211. These were subsequentlysubjected to high throughput molecular analysis to identify potentiallycorrect gene targeting events by PCR with primer pair IB527×IB616 (SEQID NO. 13 and 14 respectively, schematically represented in FIG. 3)using the Elongase enzyme mix from Invitrogen in a final MgCl2concentration of 2 mM and the following cycling parameters: 2 mindenaturation at 94° C., followed by 35 cycles of 94°-30 sec, 58°-30 sec,68°-9 min, and a final elongation step of 7 min at 68° C. The presenceof a PCR product of 9640 by is indicative of a correctly stacked genetargeting event by at least one-sided homologous recombination (via the2072 bp homology region indicated by the accolade in FIG. 3).Potentially correct stacks can also be identified by PCR with primerpair IB527×IB617 (SEQ ID NO 13 and 17 respectively,) which should thenresult in a PCR product of 3893 by (FIG. 3). Subsequent sequenceanalysis of the PCR products allows confirming at least one-sidedcorrect homologous recombination.

Since the absence of a PCR product could also result from poor DNAquality or large insertions or deletions at the target site, a number ofglyphosate resistant calli that were positive or negative on PCR werealso analyzed by southern blotting on DraIII/StuI digested genomic DNAusing probes recognizing the EPSPS gene (SEQ ID NO 9), the Cry2Ae gene(SEQ ID NO 10) and the HPPD gene (SEQ ID NO 11), respectively, understringent conditions. In case of a correct stacked event, this shouldresult in the identification of an 11 kb band using all three probes(see also FIG. 3).

In total, at least 27 putative correct stacked events have beenidentified using PCR (˜1.8% of the total of the 1479 identified glyRevents), of which at least 13 have been confirmed to be correct genetargeting events by southern blotting as described above.

Some of the confirmed stacked events were evaluated for thefunctionality of the bar and Cry2Ae gene of the original GHB119 event.Via a Leaf Strip test, expression of the PAT and Cry2Ae protein could beconfirmed in those events.

Further, sequence analysis of PCR fragment IB527×IB617 and IB589×VDS406(SEQ ID NO. 17 and SEQ ID NO. 18 respectively), which span the tworecombination sites (see FIG. 3), showed a perfect insertion of epspsand hppd at the target loci.

Example 6 Transmission of the Stack to Next Generations

In order to test whether all four transgenes are indeed transmitted tothe next generation as a stack and do not segregate independently,offspring of crosses of a plant comprising the stack with a wild-typeplants were evaluated for the presence of the transgenes.

TABLE 2 Segregation data from a cross of a targeted insertion event(i.e. heterozygous for the stacked event) with a wild type plant resultsin ca 50% WT and ca 50% 4 genes. epsps hppd cry2Ae bar WT WT WT WT 1copy 1 copy 1 copy 1 copy 1 copy 1 copy 1 copy 1 copy 1 copy 1 copy 1copy 1 copy 1 copy 1 copy 1 copy 1 copy WT WT WT WT WT WT WT WT WT WT WTWT WT WT WT WT WT WT WT WT 1 copy 1 copy 1 copy 1 copy WT WT WT WT WT WTWT WT WT WT WT WT WT WT WT WT 1 copy 1 copy 1 copy 1 copy 1 copy 1 copy1 copy 1 copy 1 copy 1 copy 1 copy 1 copy WT WT WT WT 1 copy 1 copy 1copy 1 copy 1 copy 1 copy 1 copy 1 copy 1 copy 1 copy 1 copy 1 copy WTWT WT WT WT WT WT WT

TABLE 3 Segregation data from a selfed progeny (i.e. homozygous for thestacked event) with a wild type plant results in ca 25% WT and ca 75% 4genes. epsp hppd cry2Ae bar WT WT WT WT 1 copy 1 copy 1 copy 1 copy 2copies 2 copies 2 copies 2-3 copies 1 copy 1 copy 1 copy 1 copy 1 copy 1copy 1 copy 1 copy 2 copies 2 copies 2 copies 2 copies WT WT WT WT WT WTWT WT 1 copy 1 copy 1 copy 1 copy 1 copy 1 copy 1 copy 1 copy 1 copy 1copy 1 copy 1 copy WT WT WT WT 1 copy 1 copy 1 copy 1 copy 1 copy 1 copyFailed 1 copy

Thus, table 2 and 3 show that the stacked event indeed inherits as asingle genetic unit.

Example 7 Expression and Functionality of the Transgenes in the Stack

A number of stacked events were evaluated for expression of thetransgenes using Q-PCR analysis and western blotting. Expression of allfour transgenes was observed, albeit sometimes with varying expressionlevels (which correlated between the two detection methods). Also,tolerance to HPPD-inhibitor herbicides was evaluated in the greenhousein progenies from the stacked events. The plants displayed some mildbleaching after a 2×TBT treatment but recovered afterwards.

Example 8 Generation of Cotton Plants Comprising Chimeric HPPD Genes

Using conventional recombinant DNA techniques the pTIF16 and pTSIH09T-DNA expression vector were constructed, both comprising an HPPDencoding chimeric gene, under the control of the 35S and CsVMV promoterrespectively, and an EPSPS encoding chimeric gene, with the followingoperably linked DNA fragments:

pTIF16 (SEQ ID NO. 20)

HPPD Chimeric Gene:

-   -   a) P35S2: sequence including the promoter region of the        Cauliflower Mosaic Virus 35S transcript (Odell et al., 1985): nt        position 88 to 506 of SEQ ID NO: 20    -   b) 5′cab22L: sequence including the leader sequence of the        chlorophyl a/b binding protein gene of Petunia hybrida (Harpster        et al., 1988): nt position 507-576 of SEQ ID NO: 20    -   c) TPotpY-1Pa: coding sequence of an optimized transit peptide        derivative (pos 55 changed into Tyr), containing sequence of the        RuBisCO small subunit genes of Z. mays (corn) and H. annuus        (sunflower) (Lebrun et at., 1996), adapted for cotton codon        usage: nt position 577-948 of SEQ ID NO: 20    -   d) hppdPfW336-1Pa: coding sequence of the        4-hydroxyphenylpyruvate dioxygenase gene of Pseudomonas        fluorescens strain A32 modified by the replacement of the amino        acid Glycine 336 with a Tryptophane (Boudec et al., 2001),        adapted to cotton codon usage: nt position 949-2025 of SEQ ID        NO: 20 (codon encoding tryptophan at position 336 found at nt        1954-1956)    -   e) 3′histonAt: sequence including the 3′ untranslated region of        the histone H4 gene of Arabidopsis thaliana (Chaboute et all.,        1987): nt position 2026-2714 of SEQ ID NO: 20

EPSPS Chimeric Gene:

-   -   a) Ph4a748 ABC: sequence including the promoter region of the        histone H4 gene of Arabidopsis thaliana (Chaboute et aL, 1987):        nt position 2791-3750 of SEQ ID NO: 20    -   b) intron1 h3At: first intron of gene II of the histone H3.III        variant of Arabidopsis thaliana (Chaubet et al., 1992): nt        position 3751-4229 of SEQ ID NO: 20    -   c) TPotp C: coding sequence of the optimized transit peptide        containing sequence of the RuBisCO small subunit genes of Zea        mays (corn) and Helianthus annuus (sunflower) (Lebrun et al.,        1996): nt position 4230-4601 of SEQ ID NO: 20    -   d) 2mepsps: the coding sequence of the double-mutant        5-enol-pyruvylshikimate-3-phosphate synthase gene of Zea mays        (corn) (Lebrun et aI, 1997): nt position 4602-5939 of SEQ ID NO:        20    -   e) 3′histonAt: sequence including the 3′ untranslated region of        the histone H4 gene of Arabidopsis thaliana (Chaboute et al.,        1987): nt position 5940-6630 of SEQ ID NO: 20

pTSIHO9 (SEQ ID NO. 23)

HPPD Chimeric Gene:

-   -   a) Pcsvmv XYZ: sequence including the promoter region of the        Casava Vein Mosaic Virus (Verdaguer et al., 1996): nt position        2735-2223 of SEQ ID NO: 23    -   b) TPotpY-1Pa: coding sequence of an optimized transit peptide        derivative (pos 55 changed into Tyr), containing sequence of the        RuBisCO small subunit genes of Z. mays (corn) and H. annuus        (sunflower) (Lebrun et at., 1996), adapted for cotton codon        usage: nt position 2214-1845 of SEQ ID NO: 23    -   c) hppdPfW336-1Pa: coding sequence of the        4-hydroxyphenylpyruvate dioxygenase gene of Pseudomonas        fluorescens strain A32 modified by the replacement of the amino        acid Glycine 336 with a Tryptophane (Boudec et al., 2001),        adapted to cotton codon usage: nt position 1842-766 of SEQ ID        NO: 23    -   d) 3′histonAt: sequence including the 3′ untranslated region of        the histone H4 gene of Arabidopsis thaliana (Chaboute et all.,        1987): nt position 749-83 of SEQ ID NO: 23

EPSPS Chimeric Gene:

-   -   a) Ph4a748 ABC: sequence including the promoter region of the        histone H4 gene of Arabidopsis thaliana (Chaboute et aL, 1987):        nt position 2834-3750 of SEQ ID NO: 23    -   b) intron1 h3At: first intron of gene II of the histone H3.III        variant of Arabidopsis thaliana (Chaubet et al., 1992): nt        position 3790-4255 of SEQ ID NO: 23    -   c) TPotp C: coding sequence of the optimized transit peptide        containing sequence of the RuBisCO small subunit genes of Zea        mays (corn) and Helianthus annuus (sunflower) (Lebrun et al.,        1996): nt position 4269-4640 of SEQ ID NO: 23    -   d) 2mepsps: the coding sequence of the double-mutant        5-enol-pyruvylshikimate-3-phosphate synthase gene of Zea mays        (corn) (Lebrun et aI, 1997): nt position 4641-5978 of SEQ ID NO:        23    -   e) 3′histonAt: sequence including the 3′ untranslated region of        the histone H4 gene of Arabidopsis thaliana (Chaboute et al.,        1987): nt position 5999-6665 of SEQ ID NO: 23

The T-DNA vectors pTIF16 and pTSIH09 were introduced into Agrobacteriumtumefaciens C58C1Rif (pEHA101) and transformants were selected usingspectinomycin and streptomycin according to methods known in the art.

The Agrobacterium strains were used to transform the cotton var. “Coker312” according to methods known in the art and transgenic plants wereselected in vitro for tolerance to glyphosate (1.0-1.5 mM) and analyzedfor copy number using RT-PCR. T0 plants containing the transgenes wereselfed and the resulting T1 generation was used for herbicide tolerancetests in the greenhouse.

Example 9 Assessment of Herbicide Tolerance in the Greenhouse

To analyze for herbicide tolerance, a segregating T1 population of 100seeds of a pTIF16 and a pTSIH09 event was sown in a greenhouse. Emergingplants were treated at growth stage with different HPPD inhibitors indifferent field doses. 13 and 24 DPA (days post application), plantswere scored for phenotype (“damage scores”) on a scale of 0-100, whereby0 represents no damage (i.e. corresponding to untreated wild-typeplants) and 100 represents maximum damage (i.e. displaying damage on allaerial parts) (Table 4).

TABLE 4 Damage scoring of cotton plants of the pTIF16 and the pTSIH09events comprising the chimeric Pseudomonas fluorescens (Pf)-HDDP-336Wgene, as well as wt controls (Coker312) after herbicide treatment ortreatment with the formulation without the herbicide (blind). Damagescores (% damage) Treatments (post-emergent) Coker pTIF16 pTSIH09 Dosageof WT homozygote heterozygote test average of 3 plants average of 6plants average of 10-11 plants sprayed compound Field rate per treatmentper treatment per treatment Test compound as (g/ha) equivalent 13 dpa 24dpa 13 dpa 24 dpa 13 dpa 24 dpa Blank — — 4 4 0 0 0 0 Tembotrione 200 2x65 94 6 4 18 15 400 4x 65 96 12 14 25 27 Mesotrione Callisto 200 2x 6895 11 10 23 19 (SC10) 400 4x 68 96 21 24 29 39 Topramezone Clio/ 36 2x67 97 0 1 7 9 Impact 72 4x 68 96 0 3 9 11 (SC30) Isoxaflutole Balance200 2x 50 67 2 1 6 8 (WG75) 400 4x 65 70 6 5 8 9

Example 10 ASSESSMENT of herbicide tolerance in the field

Field trials for herbicide tolerance were conducted at two locations inthe US, i.e. California (CA) and Tennessee (TN), with two replicates perlocation. Plants homozygous for the pTIF16 event in Coker 312background, along with control plants (wt segregants derived from thesame line or Coker 312 plants) were sown in plots of ca. 40 plants andtreated with a broad spectrum of HPPD inhibitor herbicides at commercialconcentrations (at least 1×) in a post-emergent treatment at the 2-4leaf stage (except for Balance Flex which was applied pre-emergence inCA and post-emergent in TN). Tolerance was evaluated by scoring forplant response (taking into account the extent of chlorosis, bleachingand necrosis) 7, 21 and 28 days after treatment (DAT) on a scale of0-100 (wherein 0 corresponds to no chlorosis/bleaching/necrosis and 100indicates death of the plant). Results were averaged for the twolocations (table 5). While wt plants displayed significant chlorosis,bleaching and necrosis in response to the herbicide treatment, plantshomozygous for the pTIF16 event were tolerant to all HPPDi herbicidestested.

TABLE 5 Plant response (chlorosis, bleaching and necrosis) of cottonplants homozygous (hom) and azygous (wt) for the pTIF16 event comprisingthe chimeric Pseudomonas fluorescens (Pf)-HDDP-336W gene. 7 DAT 21 DAT28 DAT treatment 1X a.i. wt hom wt hom wt hom Untreated — 0.0 0.0 0.00.0 0.0 0.0 Tembotrione* 100 g a.i./ha 87.8 26.3 83.8 2.8 72 1.8 LaudisSC a.i. Tembotrione, 81.3 9.5 56.8 5.5 50 0.3 138 g a.i./ha Callistoa.i. Mesotrione, 86.3 0.5 53.3 0.0 67.5 0.0 105 g a.i./ha Balance Flex**a.i. Isoxaflutole, 42.5 10 99 4 n/a n/a 105 g a.i./ha ImpactTopramezone, 92.5 7.8 62.5 1.3 83.6 0.0 18.41 g a.i./ha Pyrasulfutole37.5 g a.i./ha 83.8 0.0 58.3 3.5 62.5 0.0 *TBT treatment CA applied 1week earlier than other treatments, **Balance Flex treatment CA only

Example 11 Pre-Emengence and Post-Emergence Herbicide Tolerance in theField

Similar as above, field trials were conducted in Argentina with onepTIF16 event and two pTSHI09 events (homozygotes) in comparison with thewild type Coker 312 line. Pre-emergence tolerance was evaluated for 2×and 4× Balance Pro (IFT) and 4× Callisto (MST) at 7, 14 and 21 daysafter treatment (DAT). Post emergence tolerance was tested for 2×IFT,4×IFT, 2×MST, 2×TBT, 4×TBT 2× Topramezone (Top) and 4× glyphosate.Results (plant response) are depicted in table 6 and 7 below.

TABLE 6 Pre-emergence herbicide tolerance in the field reflected asplant response (chlorosis, bleaching and necrosis). Pre Balance Pro 2XBalance Pro 4x Callisto IFT 2x IFT 4x MST 4x DAT 7 14 21 7 14 21 7 14 21Coker 24.3 81.7 85.3 40.0 91.3 95.2 38.3 83.3 82.8 pTIF16-01 3.7 7.7 4.72.3 7.0 1.8 4.7 7.3 2.7 PTSIH09-01 1.0 4.3 0.8 2.3 0.0 0.3 3.3 3.0 0.8PTSIH09-02 2.7 2.3 1.5 2.0 3.0 1.0 4.3 5.3 4.0

TABLE 7 Post-emergence herbicide tolerance in the field reflected asplant response (chlorosis, bleaching and necrosis). Post IFT 2x IFT 4xDAT 4 7 14 24 36 4 7 14 24 36 Coker 14.7 32.8 75.8 84.5 85.3 18.3 36.768.3 81.5 80.8 PTIF16-01 2.3 2.8 1.5 4.2 5.8 1.7 1.7 1.2 1.0 0.8pTSIH09-01 0.7 3.5 1.0 0.5 2.5 2.3 0.7 0.7 0.2 0.0 pTSIH09-02 0.7 2.31.7 4.2 7.5 0.7 0.8 1.0 0.0 0.0 Meso 2x TBT 2x DAT 4 7 14 24 36 4 7 1424 36 Coker 18.0 43.0 95.3 100.0 100.0 18.0 43.7 93.3 100.0 100.0PTIF16-01 7.3 30.2 25.5 28.5 24.2 11.3 21.5 21.7 17.0 13.7 PTSIH09-017.3 36.0 28.3 31.2 26.7 8.7 23.5 22.5 19.2 16.0 PTSIH09-02 6.0 35.0 32.734.5 32.5 9.7 22.3 23.8 20.8 16.8 TBT 4x Top 4x DAT 4 7 14 24 36 4 7 1424 36 Coker 15.0 44.7 95.0 99.2 100.0 15.3 43.0 92.5 100.0 99.8PTIF16-01 5.3 19.2 20.7 13.0 11.7 7.3 17.7 22.7 16.5 12.5 PTSIH09-01 8.322.8 20.7 15.2 15.3 5.3 19.5 21.2 15.7 12.8 PTSIH09-02 5.3 23.0 21.717.0 16.7 6.3 16.8 18.5 17.5 16.3 Gly 4x DAT 4 7 14 24 36 Coker 46.793.3 99.5 99.3 100.0 PTIF16-01 0.0 0.0 0.0 0.0 0.0 pTSIH09-01 0.0 0.00.5 0.8 2.5 pTSIH09-02 0.0 0.0 0.0 0.5 2.8

1. A method for modifying the genome of a plant cell at a predefinedsite comprising: a. inducing a double stranded DNA break in the vicinityof or at said predefined site, said double stranded break being inducedby the introduction into said cell of a double stranded DNA breakinducing (DSBI) enzyme which recognizes a recognition sequence in thevicinity of or at said predefined site; and b. selecting a plant cellwherein said double stranded DNA break has been repaired resulting in amodification in the genome at said preselected site, wherein saidmodification is i. a replacement of at least one nucleotide; ii. adeletion of at least one nucleotide; iii. an insertion of at least onenucleotide; or iv. any combination of i.-iii.; wherein said predefinedsite is located in close proximity to an existing elite event. 2.(canceled)
 3. The method of claim 1, wherein said recognition sequenceis comprised within SEQ ID NO: 3 or SEQ ID NO:
 4. 4. The method of claim1, wherein said recognition sequence comprises the nucleotide sequenceof SEQ ID NO: 1 or SEQ ID NO:
 2. 5-6. (canceled)
 7. The method of claim1, wherein said DSBI enzyme is a single chain meganuclease or a pair ofmeganucleases which recognizes or recognize in concert said predefinedsite and induces or induce said double stranded break.
 8. The method ofclaim 7, wherein said meganuclease or pair of meganucleases is/arederived from I-CreI and wherein the following amino acids are present inmeganuclease unit 1: a. S at position 32; b. Y at position 33; c. Q atposition 38; d. Q at position 80; e. S at position 40; f. T at position42; g. R at position 77; h. Y at position 68; i. Q at position 70; j. Hat position 75; k. T at position 44; l. I at position 24; m. Q atposition 26; n. K at position 28; or o. N at position 30; orcombinations thereof; and wherein the following amino acids are presentin meganuclease unit 2: p. S at position 70; q. Q at position 44; r. Kat position 24; s. A at position 26; t. K at position 28; u. N atposition 30; v. S at position 32; w. Y at position 33; x. Q at position38; y. Q at position 80; z. S at position 40; aa. T at position 42; bb.Q at position 77; or cc. Y at position 68; or combinations thereof. 9.The method of claim 7, wherein said meganuclease or pair ofmeganucleases comprises the amino acid sequence of SEQ ID NO. 6 fromamino acid position 11-165 and from position 204-360.
 10. (canceled) 11.The method of claim 1, wherein prior to step b, a repair DNA molecule isdelivered into said cell, said repair DNA molecule being used as atemplate for repair of said double stranded DNA break.
 12. The methodaccording to claim 1, wherein said repair DNA comprises at least oneflanking region comprising a nucleotide sequence having sufficienthomology to the upstream or downstream DNA region of said predefinedsite to allow recombination with said upstream or downstream DNA region.13. The method according to claim 1, wherein said repair DNA comprisestwo flanking regions located on opposite ends of said repair DNA, one ofsaid flanking regions comprising a nucleotide sequence having sufficienthomology to the upstream DNA region of said predefined site, the otherflanking region comprising a nucleotide sequence having sufficienthomology to the downstream sequence of said predefined site to allowrecombination between said flanking nucleotide sequences and saidupstream and downstream DNA regions.
 14. The method according to claim1, wherein said repair DNA comprises a selectable marker gene.
 15. Themethod according to claim 1, wherein said repair DNA comprises a plantexpressible gene of interest.
 16. The method according to claim 15,wherein said plant expressible gene of interest is a herbicide tolerancegene, an insect resistance gene, a disease resistance gene, an abioticstress resistance gene, an enzyme involved in oil biosynthesis,carbohydrate biosynthesis, an enzyme involved in fiber strength or fiberlength, or an enzyme involved in biosynthesis of secondary metabolites.17. The method according to claim 16, wherein said plant expressiblegene of interest encodes a protein having HPPD activity.
 18. The methodaccording to claim 16, wherein said plant expressible gene of interestencodes a protein having HPPD activity, wherein said protein has atryptophan at a position corresponding to position 336 of SEQ ID NO: 19,and wherein said protein provides to said plant tolerance to a fielddose of at least 1× of at least one HPPD inhibitor.
 19. The methodaccording to claim 1, wherein said plant cell is further regeneratedinto a plant.
 20. The method according to claim 19, wherein said plantis further crossed with another plant. 21-36. (canceled)
 37. A plantcell comprising a modification at a predefined site of the genome,obtained by the method according to claim
 1. 38. A plant, plant part,seed or propagating material thereof, comprising a modification at apredefined site of the genome, obtained by the method according to claim19. 39-42. (canceled)
 43. A plant, plant part, seed or propagatingmaterial thereof, comprising a modification at a predefined site of thegenome, comprising the plant cells of claim 37.